Hypothalamic Membranes Research Articles

Regulation of an Opioid Receptor Chaperone Protein, RTP4, by Morphine

Signaling by classic analgesics, such as morphine, is governed primarily by the relative abundance of opioid receptors at the cell surface, and this is regulated by receptor delivery to, and retrieval from, the plasma membrane. Although retrieval mechanisms, such as receptor endocytosis, have been extensively investigated, fewer studies have explored mechanisms of receptor maturation and delivery to the plasma membrane. A previous study implicated receptor transporter proteins (RTPs) in the latter process. Since not much is known about regulation of RTP expression, we initiated studies examining the effect of chronic morphine administration on the levels of RTPs in the brain. Among the four RTPs, we detected selective and region-specific changes in RTP4 expression; RTP4 mRNA is significantly upregulated in the hypothalamus compared with other brain regions. We examined whether increased RTP4 expression impacted receptor protein levels and found a significant increase in the abundance of mu opioid receptors (MOPrs) but not other related G protein-coupled receptors (GPCRs, such as delta opioid, CB1 cannabinoid, or D2 dopamine receptors) in hypothalamic membranes from animals chronically treated with morphine. Next, we used a cell culture system to show that RTP4 expression is necessary and sufficient for regulating opioid receptor abundance at the cell surface. Interestingly, selective MOPr-mediated increase in RTP4 expression leads to increases in cell surface levels of MOPr-delta opioid receptor heteromers, and this increase is significantly attenuated by RTP4 small interfering RNA. Together, these results suggest that RTP4 expression is regulated by chronic morphine administration, and this, in turn, regulates opioid receptor cell surface levels and function.

Oral Administration of the Endocannabinoid Anandamide during Lactation: Effects on Hypothalamic Cannabinoid Type 1 Receptor and Food Intake in Adult Mice.

We have previously shown that administration of the endocannabinoid anandamide (AEA) during lactation leads to overweight, increased body fat accumulation, and insulin resistance in adult mice. This study was designed to elucidate if these effects are due to increased food intake, stimulated by an augmented abundance and binding ability of the hypothalamic cannabinoid type 1 receptor (CB1R). With this aim, male mice pups were treated with a daily oral dose of AEA during lactation. Adult mice were also treated with a single oral dose of AEA, to evaluate acute food intake during 4 h. At 21 and 160 days, CB1R protein abundance was calculated by western blot analysis. Capacity of hypothalamic membranes to specifically bind the radioligand 3[H]-CP55.940 was also measured. Western blots showed a 72% increase in CB1R abundance in AEA-treated 21-day-old mice, without differences in adult mice. Additionally, specific binding of 3[H]-CP55.940 to hypothalamic membranes from adult mice was significantly lower in those mice treated with AEA during lactation. Moreover, AEA did not stimulate acute food intake in both, AEA-treated and control mice. Results suggest that metabolic alterations found in adult mice because of AEA treatment during lactation are not associated with hypothalamic CB1R.

Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus

The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5′triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,β-methyleneadenosine-5′triphosphate (α,β-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5–10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,β-meATP to displace [α-35S]deoxyadenosine-5′thiotriphosphate ([35S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5′diphosphate (2-MeSADP) activated 75–85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the high potency to dose-dependently stimulate ARC cells of the LDD group, the high affinity for rat P2X(1–3) and low affinity for rat P2X4, P2X7 and P2Y receptor subtypes except P2Y1 and P2Y13, the agonist 2-MeSATP primarily acted upon P2X2 and P2Y1 purinoceptors to trigger intracellular calcium signaling in ARC neurons and astrocytes.

Molecularly imprinted polymer microprobes for manipulating neurological function by regulating temperature-dependent molecular interactions

We describe molecularly imprinted polymer (MIP) microprobes for manipulating neurological function by the thermoregulatory process. Some target and disease areas show raised temperature than normal temperature which are involved in neuronal diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases. As well thermodynamic parameters can be obtained in the environments in vitro, which that the assay involves a temperature variable, at or beyond body temperature. We applied for studying molecular aspects of hypothalamus regulated physiology and behaviors. Steady-state binding was detected by fluorescence and Boltzmann energies-based fluorescence emission showed interactions of clozapine with altered energy levels. Dynamic spectra revealed dramatic effects on the fluorescence of the drug in dopamine imprint of MIPs exposed rat hypothalamic membranes at different temperatures. The MIPs that cooperatively occupied and provided the response to the neural proteins were not likely to be involved in the binding of clozapine within the cavity. The results indicated that the dynamics on the surface of proteins exposed to the drug–MIP with assembly, mediated specific function through temperature-dependent molecular interaction in the hypothalamus. Thus, this approach is applicable to processes of key importance of controlling the energy homeostasis and, plausibly, to selective signaling can affect in plasticity of the neural response.

A Novel Path to Obesity

A Novel Path to Obesity

Functional rundown of gamma‐aminobutyric acidAreceptors in human hypothalamic hamartomas

Human hypothalamic hamartomas (HHs) are highly associated with treatment-resistant gelastic seizures. HHs are intrinsically epileptogenic, although the basic cellular mechanisms responsible for seizure activity are unknown. Altered gamma-aminobutyric acid (GABA) function can contribute to epileptogenesis in humans and animal models. Recently, functional GABA(A) receptor (GABA(A) R) rundown has been described in surgically resected human temporal lobe epilepsy tissue. We asked whether functional GABA(A) R rundown also occurs in human HH neurons. GABA(A) R-mediated currents were measured using perforated patch-clamp recordings in single neurons acutely dissociated from surgically resected HH tissue. In addition, functional GABA(A) Rs were expressed in Xenopus oocytes after microinjection with membrane fractions from either HH or control hypothalamus, and were studied with 2-electrode voltage-clamp recordings. Perforated patch-clamp recordings in dissociated HH neurons showed that repetitive exposure to GABA (5 consecutive exposures to 0.1 mM GABA with 1-second duration and at 20-second intervals) induced a time-dependent rundown of whole-cell currents in small HH neurons, whereas large HH neurons showed much less rundown using the same protocol. Functional rundown was not observed in HH neurons with repetitive exposure to glycine or glutamate. Two-electrode voltage-clamp recordings (6 consecutive exposures to 1 mM GABA with 10-second duration and at 40-second intervals) induced GABA current rundown in Xenopus oocytes microinjected with HH membrane proteins, but not in the oocytes expressing hypothalamic membrane proteins derived from human autopsy controls. Functional rundown of GABA currents was significantly attenuated by intracellular application of adenosine triphosphate or the nonspecific phosphatase inhibitor, okadaic acid. Neurons from surgically resected human HH demonstrate functional rundown of GABA(A) R-mediated transmembrane currents in response to GABA agonist exposure. Rundown may be a marker for impaired GABAergic function and a contributing mechanism for seizure genesis within HH tissue.

Microsurgical anatomy of Liliequist’s membrane demonstrating three-dimensional configuration

Liliequist's membrane (LM) is an important arachnoid structure in the basal cisterns. The relevant anatomic descriptions of this membrane and how many leaves it has are still controversial. The existing anatomical theories do not satisfy the needs of minimally invasive neurosurgery. We aimed to establish the three-dimensional configuration of LM. Fifteen adult formalin-fixed cadaver heads were dissected under a surgical microscope to carefully observe the arachnoid mater in the suprasellar and post-sellar areas and to investigate the arachnoid structure and its surrounding attachments. It was found that the LM actually consists of three types of membranes. The diencephalic membrane (DM) was usually attached by the mesencephalic membrane (MM) from underneath, and above DM it was usually a pair of hypothalamic membranes (HMs) extending superomedially. The pair of HMs was stretched between the DM (or MM) and the hypothalamus and were seldom attached to the carotid-chiasmatic walls between the carotid cistern and the chiasmatic cistern. These three types of membranes (DM, MM, and HM) comprised the main arachnoid structure in the anterior incisural space and often presented as four connected leaves. However, only two thirds of the specimens had all three types of membranes, and there was considerable variation in the characteristics and shapes of the membranes among the specimens. All three types of membranes comprising LM serve as important anatomical landmarks and interfaces for surgical procedures in this area.

Brain AT1Angiotensin Receptor Subtype Binding: Importance of Peptidase Inhibition for Identification of Angiotensin II as Its Endogenous Ligand

The existence and localization of brain angiotensin receptors is well established. However, questions regarding the endogenous ligand for brain angiotensin type 1 (AT(1)) receptors necessitates re-examination of brain angiotensin receptor binding studies. To assess the ability of angiotensin II to bind to the brain AT(1) receptor, radioligand binding studies of rat brain AT(1) receptors were performed using both (125)I-angiotensin II and (125)I-sarcosine(1), isoleucine(8) angiotensin II. Determination of binding kinetics and competition by an AT(1) receptor antagonist was carried out to reveal the identity of the membrane binding sites and to identify the bound (125)I-labeled molecules. Initial analysis of (125)I-angiotensin II binding to hypothalamic membranes using an established protocol revealed that a negligible amount of intact radioligand was bound to the membranes. In contrast, binding of (125)I-sarcosine(1), isoleucine(8) angiotensin II was saturable, of high affinity, and primarily as intact radioligand. Sequential addition of four peptidase inhibitors-o-phenanthroline, puromycin, phenymethylsulfonyl fluoride, and glutamate phosphonate-to the assay buffer dramatically increased the binding of (125)I-angiotensin II to rat brain membranes: more than 75% of the bound (125)I was the intact radioligand, and the binding was of high affinity and saturable. Some, but not all, of the binding could be displaced by the AT(1)-selective antagonist losartan. This demonstrates that (125)I-angiotensin II can bind to brain AT(1) receptors and does not require conversion to (125)I-angiotensin III to bind to brain AT(1) receptors.

Adiponectin receptors are expressed in hypothalamus and colocalized with proopiomelanocortin and neuropeptide Y in rodent arcuate neurons

Adiponectin is involved in the control of energy homeostasis in peripheral tissues through Adipor1 and Adipor2 receptors. An increasing amount of evidence suggests that this adipocyte-secreted hormone may also act at the hypothalamic level to control energy homeostasis. In the present study, we observed the gene and protein expressions of Adipor1 and Adipor2 in rat hypothalamus using different approaches. By immunohistochemistry, Adipor1 expression was ubiquitous in the rat brain. By contrast, Adipor2 expression was more limited to specific brain areas such as hypothalamus, cortex, and hippocampus. In arcuate and paraventricular hypothalamic nuclei, Adipor1, and Adipor2 were expressed by neurons and astrocytes. Furthermore, using transgenic green fluorescent protein mice, we showed that Adipor1 and Adipor2 were present in pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) neurons in the arcuate nucleus. Finally, adiponectin treatment by intracerebroventricular injection induced AMP-activated protein kinase (AMPK) phosphorylation in the rat hypothalamus. This was confirmed by in vitro studies using hypothalamic membrane fractions. In conclusion, Adipor1 and Adipor2 are both expressed by neurons (including POMC and NPY neurons) and astrocytes in the rat hypothalamic nuclei. Adiponectin is able to increase AMPK phosphorylation in the rat hypothalamus. These data reinforced a potential role of adiponectin and its hypothalamic receptors in the control of energy homeostasis.

A thermodynamic model of the action–perception cycle in brain dynamics

We previously demonstrated that in vivo estradiol treatment markedly attenuatesα2-adrenoceptor function and coupling to G-proteins in the hypothalamus of female rats. Ligand binding studies indicated that 48 h exposure to estradiol decreases the number ofα2-adrenergic receptors in the agonist high affinity state. In the present studies, when [3H]RX821002 was used to label brainα2-adrenoceptors, the density of binding sites significantly increased in the hypothalamus and preoptic area 48 h after estrogen treatment. Moreover, the thermodynamics of ligand binding toα2-adrenergic receptors in membranes of female rat hypothalamus were modified by the same estradiol treatments that reduceα2-adrenoceptor function. In hypothalamic membranes from ovariectomized control rats, antagonist (RX821002)-receptor binding was primarily entropy-driven while agonist (oxymetazoline) binding had a higher enthalpy component. In membranes from estradiol-exposed animals, the entropic contribution to both agonist and antagonist binding was markedly increased, and the enthalpy component was reduced. Since the thermodynamic characteristics of ligand-receptor binding are strongly correlated with efficacy in activating signal transduction [36], these data raise the intriguing possibility that steroids regulate transmembrane signaling by stabilization of a receptor conformation with reduced intrinsic efficacy.

Inhibition of proteolytic degradation and characterization of binding of natural angiotensins to AT1 receptors in brain and liver

Metabolism of angiotensins in receptor binding assays is a continuing problem. The suggestion that brain AT1 receptors (AT1R) differ from peripheral AT1R necessitates accurate appraisal of brain AT1R binding. The goal of this study was to establish experimental conditions that preserve 125I‐Ang II and ‐III, to characterize their binding to rat hypothalamic and liver AT1R. Negligible specific binding of 125I‐Ang III and ‐II is observed in hypothalamic membranes (45 or 60min at 24oC in 5mM EDTA, 150mM NaCl, 0.1mM bacitracin, 50mM NaPO4, pH 7.2 buffer + 10 μM PD123319) due to severe proteolytic degradation, as < 5% of bound 125iodine corresponds to 125I‐Ang III or ‐II. In liver membranes, 125I‐Ang III is severely degraded, whereas 125I‐Ang II shows high affinity saturable binding to AT1R with > 50% of bound 125I corresponding to 125I‐Ang II. Addition of 1 mM o‐phenanthroline & phenylmethylsulfonyl fluoride, and 3 mM puromycin & glutamate phosphonate to the assay buffer increased bound 125I‐Ang II and ‐III to > 80%, both in liver and hypothalamic membranes revealing abundant, high‐affinity binding to AT1R. The affinity of both ligands for brain AT1R was less than for liver. 125I‐Ang III showed a smaller Bmax for AT1R in liver than 125I‐Ang II. These results indicate that both peptides bind with higher affinity for liver AT1R than brain AT1R, but that 125I‐Ang II binds to a larger population of AT1R in liver than does 125I‐Ang III.

Evidence for a Role of the GHS‐R1a Receptors in Ghrelin Inhibition of Gastric Acid Secretion in the Rat

Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R) has been previously shown to inhibit gastric acid secretion in pylorus-ligated rats. Two isoforms of GHS-R have been identified: GHS-R(1a) and GHS-R(1b). The present study aimed: (i) to characterise the type of GHS-R involved in the central gastric inhibitory activity of ghrelin by using des-octanoyl ghrelin, and synthetic GHS-R(1a) agonist (EP1572) and antagonist (D-Lys(3)-GHRP-6) and (ii) to investigate the relationship between ghrelin and cortistatin (CST) in the control of gastric acid secretion by using the natural neuropeptide CST-14 and the synthetic octapeptide CST-8. The specific interactions of all the compounds with GHS-R(1a) were determined by comparing their ability to displace labelled ghrelin or somatostatin from its receptors on rat hypothalamic membranes or on rat cardiomyocyte, respectively. Intracerebroventricular administration of 0.01 and 1 nmol/rat des-octanoyl ghrelin did not affect gastric acid secretion in pylorus-ligated rats, whereas EP1572 either i.c.v. (0.01-1 nmol/rat) or i.p. (10 and 20 nmol/kg) inhibited acid gastric secretion. Preteatment with D-Lys(3)GHRP-6 (3 nmol/rat, i.c.v.) was able to remove the inhibitory action of ghrelin (0.01 nmol/rat, i.c.v.) on gastric acid volume and acid output, thus indicating that the type 1a GHS-R likely mediates the gastric inhibitory action of ghrelin. This is supported by binding data showing that D-Lys(3)GHRP-6, but not des-octanoyl ghrelin, binds to hypothalamic GHS-R. CST-14 (1 nmol/rat, i.c.v.) did not affect either basal or ghrelin inhibition of gastric acid secretion. CST-8 (1 nmol/rat, i.c.v.) was able to counteract the gastric ghrelin response. The observation that CST-14 binds both GHR-S and somatostatin receptors, whereas CST-8 specifically displaces only ghrelin binding, indicates that CST-8 behaves as a GHS-R(1a) antagonist.

Expression of gamma-aminobutyric acid B receptor subunits in hypothalamus of male and female developing rats

GABA and its receptors show particular ontogenic distributions in different rat brain areas. Recently, GABA B receptors (GBR) have been described to assemble as heterodimers formed by a GBR1a/b and a GBR2 subunit. Here, the ontogeny of rat GBRs and the pattern of subunit expression in both sexes were determined in the hypothalamus, a critical area for homeostatic regulation. Male and female rats were sacrificed at 1, 4, 12, 20, 28, 38 days of life and at adulthood and hypothalami were removed and frozen. Western blots analysis for GBR1 and GBR2 subunits showed that both were expressed in male and female hypothalamic membranes from day 1 to adulthood. In females, both GBR1a and GBR1b were maximally expressed in newborns and decreased towards adulthood. At birth, expression of GBR1a was significantly higher than GBR1b, while at 38 days, GBR1b was more abundant. In males, GBR1a and GBR1b expression was higher in young animals and decreased gradually showing adult levels between the second and third weeks of age without differences between isoforms. Comparing GBR1 variants levels in hypothalamus between sexes, GBR1a was significantly more abundant in females at birth while at 38 days its expression was higher in males; GBR1b showed no sex differences along development. GBR2 was detected in hypothalami of females and males at all ages; maximum levels were observed at 12 days and adult levels were attained at 38 days, without sex differences. This is the first report on the ontogeny of hypothalamic GABA B receptors in male and female rats, with a particular developmental pattern of subunit and isoform expression and presenting some sex differences.

Sarcosine 1,glycine 8 angiotensin II is an AT 1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT 1 and AT 2, revealed anomalous characteristics of sarcosine 1,glycine 8 Ang II (Sar 1,Gly 8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT 2 receptors, while dipsogenic responses to Ang II are mediated by AT 1 receptors, suggests that Sar 1,Gly 8 Ang II is AT 1 selective. Sar 1,Gly 8 Ang II competed for 125I-sarcosine 1,isoleucine 8 Ang II binding to AT 1 receptors in pituitary, liver and adrenal (the latter with the AT 2 selective antagonist PD 123,319) with K i's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the K i of Sar 1,Gly 8 Ang II for AT 2 receptors in rat adrenal (with the selective AT 1 antagonist losartan) was 52 nM. 125I-Sar 1,Gly 8 Ang II (0.5–3 nM) bound to AT 1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity ( K d=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT 2 receptor. 125I-Sar 1,Gly 8 Ang II selectively labeled AT 1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar 1,Gly 8 Ang II to be the first angiotensin peptide analog to show AT 1 receptor selectivity. 125I-Sar 1,Gly 8 Ang II offers a new means to selectively radiolabel AT 1 receptors and may help to characterize ligand docking sites and agonist switches for AT 1 versus AT 2 receptors.

DR4004, a putative 5-HT7 receptor antagonist, also has functional activity at the dopamine D2 receptor

The tetrahydrobenzindole, 2a-(4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl)-2a,3,4,5-tetrahydrobenzo[cd]indol-2(1H)-one (DR4004) has been described as a highly selective antagonist for the 5-hydroxytryptamine7 (5-HT7) receptor [J. Med. Chem. 42 (1999) 533]. Consistent with original data, DR4004 bound to rat hypothalamic membranes with an affinity of 7.3±0.2 (pKi±S.E.M.) for the 5-HT7 receptor. However, competition binding studies showed that DR4004 had poor receptor selectivity with the following affinity profile; dopamine D2 receptor, α1-adrenoceptor ≥5-HT7 receptor>histamine H1 receptor, α2-adrenoceptor>dopamine D1 receptor>β-adrenoceptor, muscarinic and 5-HT2A/C receptors. In conscious rats DR4004 (1, 5 or 10 mg/kg i.p.) produced a dose-dependent hyperglycaemia and hypothermia, but the former was reduced by the dopamine D2 receptor antagonist raclopride. Another 5-HT7 receptor antagonist, (R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol (SB-269970) produced hypothermia but no hyperglycaemia. This study confirms that DR4004 has high affinity for the 5-HT7 receptor but suggests that dopamine D2 receptor activity contributes to some of the in vivo effects.

Changes in histamine H3 receptor responsiveness in mouse brain.

Changes in various histamine (HA) H3 receptor-mediated responses and H3 receptor binding in brain were investigated in mice receiving single or repeated administration of ciproxifan, a potent brain-penetrating and selective H3 receptor antagonist. Blockade of the H3 autoreceptor was nearly as effective in enhancing levels of tele-methylhistamine (t-MeHA), a major HA metabolite, in brain areas when ciproxifan was administered once either at 7 a.m. or 8 p.m., in spite of the large differences of basal levels at these two phases of the circadian cycle. Blockade after a single ciproxifan administration was, however, followed by a transient decrease in striatal t-MeHA levels, possibly reflecting rapid development of autoreceptor hypersensitivity. Following a 5-day administration of ciproxifan and a 2-day drug-free period, basal t-MeHA levels were significantly decreased (approximately -20%) in three brain areas, and the ED50 values of the drug to enhance t-MeHA levels were increased by 5-15 times without significant change in maximal response, indicating that H3 autoreceptor hypersensitivity had developed. However, in synaptosomes from the cerebral cortex of these animals, the H3 receptor-mediated inhibition of K+-induced [3H]HA release was not significantly modified. Subchronic administration of ciproxifan for 10 days also resulted in an increased binding of [125I]iodoproxyfan to the H3 receptor of striatal and hypothalamic membranes by 40-54%. Hypersensitivity at H3 somatodendritic autoreceptors and at heteroreceptors attributable to an increased number of HA binding sites could account for the various changes observed in this study.

Cancer anorexia and cachexia

Patients with cancer cachexia experience a profound wasting of adipose tissue and lean body mass. Anorexia, although often present, is insufficient to account for tissue wasting because 1) cachexia involves massive depletion of skeletal muscle that does not occur during anorexia, 2) nutritional supplementation cannot replenish the loss of lean body mass, 3) cachexia can occur without anorexia, and 4) food intake might be normal for the lower weight of the cancer patient. Anorexia can arise from 1) decreased taste and smell of food, 2) early satiety, 3) dysfunctional hypothalamic membrane adenylate cyclase, 4) increased brain tryptophan, and 5) cytokine production. Appetite stimulants such as cyproheptadine, medroxyprogesterone acetate, and megestrol acetate do not significantly improve lean body mass. Tumor products might be more important in the development of cachexia. Cachectic patients excrete in their urine a lipid-mobilizing factor that directly stimulates lipolysis in a cyclic AMP–dependent manner and increases energy expenditure. Loss of skeletal muscle in cachexia is caused by upregulation of the ubiquitin–proteasome catabolic pathway. Cachexia-inducing tumors elaborate a sulfated glycoprotein, which directly initiates protein catabolism in skeletal muscle. The action of this proteolysis-inducing factor is attenuated by the polyunsaturated fatty acid eicosapentaenoic acid, which is also effective in preventing loss of skeletal muscle in cancer patients. Antagonists of tumor catabolic factors will provide important new agents in the treatment of cancer cachexia.

Binding of 125I-labeled ghrelin to membranes from human hypothalamus and pituitary gland.

Ghrelin has been proposed as a natural ligand of the GH secretagogue receptor(s) (GHS-R), which was an orphan receptor activated by synthetic peptidyl (hexarelin) and non-peptidyl (MK-0677) GHS to strongly release GH in animals and humans. Herein we studied: 1) the binding of 125I-labeled human ghrelin to membranes from human hypothalamus and pituitary gland; 2) the ability of human ghrelin (either octanoylated or desoctanoylated), as well as of some GHS and neuropeptides to compete with the radioligand. The saturation binding analysis showed, in both tissues, the existence of a single class of high-affinity binding sites with limited binding capacity. The Bmax (maximal number of binding sites) values of ghrelin receptors in the hypothalamus were significantly greater (p<0.001) than those detected in the pituitary, whereas the Kd (dissociation constant) values in the two tissues were similar. 125I-ghrelin bound to hypothalamic membranes was displaced by ghrelin, hexarelin, MK-0677, various GHS antagonists (EP-80317, [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P) and some natural (cortistatin-14) and synthetic (vapreotide) SRIH-14 agonists. In contrast, no competition was seen in the presence of GHRH-44, SRIH-14 or desoctanoylated ghrelin, a ghrelin precursor that is devoid of GH-releasing properties. In conclusion, this preliminary study firstly demonstrates that ghrelin needs octanoylation to bind its hypothalamo-pituitary receptors. These receptors are the specific binding sites for GHS and their antagonists, as well as for SRIH analogs (vapreotide and cortistatin- 14), but not for native SRIH.

The effect of melanotropic peptides on binding of [ 3H]dihydroalprenolol-hydrochloride to hypothalamic membranes

In the present work we studied the interaction of α-melanocyte-stimulating hormone (α-MSH) and ACTH-(1–24) with β-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [ 3H]dihydroalprenolol-hydrochloride ([ 3H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by α-MSH or ACTH-(1–24). These data indicate a non competitive interaction between these melanocortin peptides and [ 3H]DHA on β-adrenergic receptors in hypothalamic membranes.

Hypothalamic GABA receptor functional regulation and liver cell proliferation.

GABAergic alterations in hypothalamus during compensatory hyperplasia after partial hepatectomy (PH), lead nitrate (LN) induced direct hyperplasia and N-nitrosodiethylamine (NDEA) induced neoplasia in liver were investigated. Serum GABA levels were increased in all 3 experimental groups compared with the control. GABA content decreased in hypothalamus of PH and NDEA treated rats, while it increased in LN treated rats. GABA(A) receptor number and affinity in hypothalamic membrane preparations of rats showed a significant decrease in PH and NDEA treated rats, while in LN treated rats the affinity increased without any change in the receptor number. The GABA(B) receptor number increased in PH and NDEA treated rats, while it decreased in LN treated rats. The affinity of the receptor also increased in NDEA treated rats. Plasma NE levels showed significant increase in PH and NDEA rats compared with the control while it decreased in LN treated rats. The results of the present study suggests that liver cell proliferation is influencing the hypothalamic GABAergic neurotransmission and these changes regulate the hepatic proliferation through the sympathetic stimulation.