Expression profile of the copper homeostasis gene, rAtox1, in the rat brain.

In humans the regulation of cellular copper homeostasis is essential for proper organ development and function. A novel cytosolic protein, named Atox 1, was recently identified in yeast that functions in shuttling intracellular mononuclear copper [Cu(I)] to copper-requiring proteins. Atox 1 and its human homolog, hAtox1, are members of an emerging family of proteins termed copper chaperones that are involved in the maintenance of copper homeostasis. Northern blot analysis demonstrates that Atox 1 is widely expressed at varying levels in a variety of rat tissues including brain. Using in situ hybridization histochemistry, we characterized the expression profile for the rat homolog of Atox1 (rAtox1) in the normal adult rat brain. There is widespread expression within the brain that appears to be primarily neuronal. The highest levels of Atox1 message consists of distinct neuronal subtypes that are also characterized by their high levels of metals like copper, iron, and zinc, which include the pyramidal neurons of the cerebral cortex and hippocampus in addition to the neurons of the locus coeruleus. The high levels of a metal chaperone like Atox1 in subsets of neurons that also sequester metals suggests that Atox1 may be important in maintaining the functionality of metal requiring enzymes. A detailed analysis of the restricted expression profile for a novel copper chaperone, rAtox1, is described in the adult rat CNS. Further analysis shows that Atoxl expression is associated with neuronal populations that sequester copper.

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland.

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.

Functional corticotropin-releasing factor receptors in human neuroblastoma cells.

The present study examined the presence of functional corticotropin-releasing factor (CRF) receptors in IMR-32 neuroblastoma cells. [125I]Tyro-ovine CRF binding was linear with increasing protein concentrations, saturable, reversible and of high affinity. Scatchard analysis indicated a Kd of approximately 0.8 nM and a Bmax of approximately 32 fmol/mg protein. Competition studies with CRF and related peptides revealed a pharmacological profile characteristic of the CRF1 receptor subtype. CRF stimulated cAMP production in a dose-dependent manner with an apparent EC50 of approximately 4 nM. In addition, the putative CRF receptor antagonist alpha-helical CRF9-41 dose-dependently inhibited CRF stimulated (10 nM) cAMP production with an IC50 of approximately 60 nM. CRF treatment down regulated its own receptor while treatment with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), increased CRF binding in neuroblastoma cells. Taken together, these data demonstrate the utility of the human neuroblastoma cell line for functional studies on CRF receptors and suggest that CRF may play a regulatory role in the pathophysiology of human neuroblastoma.

Effect of aging on concentrations of D2-receptor-containing neurons in the rat striatum.

Striatal D2 dopamine receptor concentrations were shown to decrease 30-35% during the lifespan of Wistar rats as assessed both radiochemically and autoradiographically. Binding densities and degree of age-change varied within the striatum; the latter ranging from 17 to 44% in 4 different regions. Overall neuronal loss during aging was 19%, and also varied considerably within the different striatal regions. Thus, it appears that neuronal loss may account for up to roughly half of the striatal D2 receptor loss during aging.

Corticotropin-releasing factor as a transmitter in the human olivocerebellar pathway.

This study demonstrates that the neuropeptide, corticotropin-releasing factor (CRF), is present in neurons of the human inferior olivary complex (IOC). The medulla (including the inferior olive) and the anterior vermis of the cerebellum of 6 human controls obtained at autopsy were immunostained with an antibody directed against CRF. CRF receptors in cerebellum were localized with labeled CRF using in vitro receptor autoradiography. The great majority of neurons in all divisions of the IOC expressed CRF immunoreactivity, and CRF-immunoreactive fibers were demonstrated in the hilus of the olive and in the molecular layer of the cerebellum, where they closely resembled climbing fibers as visualized with other methods. CRF receptors were enriched in the cerebellum, with the highest density in inner portions of the molecular layer. These findings in human brain, consistent with studies in tissues from rat, cat, and monkey, demonstrate that CRF may be a peptidergic transmitter in the IOC climbing fiber system and that CRF receptors are expressed by cellular targets in the cerebellum.

Monoamine oxidase inhibitors inhibit [3H]quinpirole binding in rat striatal membranes.

This study describes interactions of monoamine oxidase inhibitors at binding sites labeled by [3H]quinpirole, a putatively selective ligand for dopamine D2-like receptors, in in vitro binding assays in rat brain. Monoamine oxidase inhibitors potently and competitively inhibited equilibrium binding of [3H]quinpirole in homogenate binding assays with the following rank order of potencies: clorgyline > or = Ro 41-1049 > pargyline > (-)-deprenyl > (+)-deprenyl > Ro 16-6491 > iproniazid. This rank order of potencies does not correlate with the potencies of these drugs at monoamine oxidase-A or monoamine oxidase-B, sigma site(s) or dopamine receptors. Monoamine oxidase inhibitors did not alter the ability of quinpirole to compete for [3H]spiperone binding. Quinpirole did not inhibit monoamine oxidase-A or monoamine oxidase-B activity and had low affinity (200 nM) for sigma site(s). These data suggest a potential novel binding site for [3H]quinpirole in rat brain and/or an alternative site of action for the antidepressant effects of monoamine oxidase inhibitors.

Impairment of albuterol-induced suppression of food intake in diabetes mellitus.

Albuterol (salbutamol), a beta 2 adrenoreceptor agonist, produced a dose-dependent decrease in food intake in Sprague-Dawley male control rats. This phenomenon appeared to be impaired in streptozotocin (STZ) diabetic rats. The density of beta 2 adrenoreceptors in the ventromedial hypothalamic nucleus was increased as a function of diabetes. In contrast, a decrease in the ventromedial hypothalamic 5-hydroxyindoleacetic acid (5-HIAA) concentration, an indicator of serotonin (5-hydroxytryptamine; 5-HT) release or turnover rate, was observed in this disease state. Neither the beta 2 adrenoreceptor level nor 5-HT turnover rate was altered in the periventricular hypothalamic nucleus of STZ diabetic rats. The concentrations of 5-HT in both hypothalamic nuclei were unchanged in these animals. Neurochemical and behavioral abnormalities featured in the diabetic state were reversed with institution of insulin therapy. These data conclude that diabetes-related impairment in the anorexic action of albuterol may be due to derangements in ventromedial hypothalamic beta 2 adrenoreceptor function.

Diabetes-related changes in brain beta adrenoreceptors in rats as assessed by quantitative autoradiography: relationship to hypothalamic norepinephrine metabolism and pituitary-gonadal hormone secretion.

Streptozotocin (STZ)-induced diabetes produced significant and selective alterations in brain beta adrenoreceptor subtypes, norepinephrine (NE) metabolism and pituitary-testicular hormone in male rats. The densities of beta-1 but not beta-2 adrenoreceptors were increased in hypothalamus (anterior and lateral nuclei), thalamus (ventral posterior nucleus) and amygdala (basolateral nucleus) of the STZ diabetic rats. In contrast, a decrease in the rate of metabolism of NE (estimated by the concentration of the NE metabolite, 3-methoxy-4-hydroxyphenylglycol) was observed in these STZ-treated animals. Serum concentrations of luteinizing hormone and testosterone were lower in the STZ diabetic rats. Pretreatment of rats with nicotinamide prevented the induction of hyperglycemia, upregulation of brain beta-1 adrenoreceptors, decreases in NE metabolism and reduction in serum levels of luteinizing hormone and testosterone seen in STZ-treated rats. These data suggest that derangements in the hypothalamic-pituitary-testicular axis seen in uncontrolled diabetes may be secondary to decreases in NE metabolism and compensatory upregulation of beta-1 adrenoreceptors in brain.

Differential pharmacological profile of striatal and cerebellar dopamine receptors labeled by [3H]quinpirole: identification of a discrete population of putative D3 receptors.

We have previously identified [3H]quinpirole-labeled dopamine receptors in the molecular layer of cerebellar lobule 10 which have a D2-like pharmacological profile, are guanine nucleotide-insensitive, and are juxtaposed to putative D3 receptor mRNA. This study compares the pharmacological profiles of [3H]quinpirole-labeled dopamine receptors in striatum and cerebellar lobule 10 using quantitative autoradiography. Dopaminergic compounds inhibited the specific binding of [3H]quinpirole in the caudate/putamen with the following rank order of potencies: spiperone > haloperidol > or = (+)butaclamol > or = quinpirole > or = 7-OH-DPAT > or = bromocriptine > clozapine > (-)sulpiride. In cerebellar lobule 10, a somewhat different rank order of potencies was observed: 7-OH-DPAT > quinpirole > or = bromocriptine > spiperone > (+)butaclamol > haloperidol > clozapine > (-)sulpiride. Quinpirole possessed equal affinity for [3H]quinpirole-labeled receptors in the caudate/putamen and cerebellum. 7-OH-DPAT exhibited 5-fold greater affinity for cerebellar receptors than those in the caudate/putamen. Spiperone, haloperidol, (+)butaclamol, and clozapine were more potent in competing for [3H]quinpirole binding at striatal dopamine receptors than cerebellar receptors by 83-, 59-, 11-, and 6-fold, respectively. The relative potencies of these compounds at striatal and cerebellar dopamine receptors are generally similar to the differential affinities reported at D2 and D3 dopamine receptors expressed in CHO cells, respectively. These data provide additional evidence that the dopamine receptors observed in cerebellar lobule 10 represent a discrete population of putative D3 receptors.

Characterization of [3H]quinpirole binding to D2-like dopamine receptors in rat brain.

The putative D2 dopamine receptor agonist quinpirole (LY 171,555) is the most widely used D2 agonist in in vivo and in vitro studies of D2 receptor-mediated effects. In addition, quinpirole may have even higher affinity for the recently described D3 dopamine receptor. The present study describes the in vitro binding properties of newly developed [3H]quinpirole in rat brain. [3H]Quinpirole binding was characterized in striatal membrane homogenate preparations using a filtration assay. Nonspecific binding was defined by 1 microM (+)-butaclamol. Specific [3H]quinpirole binding was saturable, and dependent on temperature, membrane concentration, sodium concentration and guanine nucleotides. Saturation analysis revealed high affinity binding characteristics (KD = 2.3 +/- 0.3 nM) which were confirmed by association-dissociation kinetics. The pharmacological profile of [3H]quinpirole binding in striatum was: (-)-N-n-propylnorapomorphine (+/-)-2-amino-6,7-dihydroxyl-1,2,3,4-tetrahydronaphthalene greater than or equal to quinpirole greater than apomorphine greater than bromocriptine greater than dopamine greater than SKF 38393 much greater than 5-hydroxytryptamine for putative dopamine agonists; spiperone greater than (+)-butaclamol greater than haloperidol greater than (-)-sulpiride greater than clozapine greater than SCH 23390 much greater than cinanserin for antagonists. [3H]Quinpirole binding exhibited stereoselectivity: (-)-sulpiride greater than (+)-sulpiride and (+)-butaclamol greater than (-)-butaclamol. This pharmacological profile is similar, though-not identical, to that observed for [3H] spiperone-labeled D2 receptors. The regional distribution of [3H]quinpirole binding sites roughly paralleled the distribution of [3H]spiperone binding sites, with greatest densities present in the striatum, nucleus accumbens and olfactory tubercles.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-dependent reduction in hepatic and splenic contents of interleukin-1 beta in experimental diabetes.

Interleukin-1 beta (IL-1 beta) is a polypeptide produced by a variety of cells of hematological, dermal and neural origin. We have investigated the effect of type I diabetes mellitus and insulin treatment on tissue levels of IL-1 beta using streptozotocin (STZ)-treated mouse as an animal model. Diabetes affected IL-1 beta in a tissue specific manner. For example, IL-1 beta levels (as measured by ELISA) were markedly decreased in the liver and spleen of the STZ diabetic mice. In contrast, the levels of this cytokine remained unalatered in other tissues including kidney, testis, hippocampus and pituitary. Insulin treatment restored the diabetes-related decreases in liver and spleen IL-1 beta levels. Overall, the present data suggest that the abnormalities in hepatic and splenic IL-1 beta levels may contribute, at least in part, to the immunodeficiency and increased susceptibility to infection in diabetes mellitus.

[3H]quinpirole binding to putative D2 and D3 dopamine receptors in rat brain and pituitary gland: a quantitative autoradiographic study.

The putative D2 dopamine receptor agonist quinpirole (LY 171,555) has been extensively used in a variety of in vivo and in vitro studies of D2 receptor-mediated effects and may have even higher affinity for the recently described D3 dopamine receptor. In the present study, conditions for autoradiographic visualization of [3H]quinpirole-labeled D2-like dopamine receptors were optimized and binding to slide-mounted sections was characterized with respect to pharmacology, guanine nucleotide sensitivity and regional distribution. The pharmacological profile of [3H]quinpirole binding in slide-mounted brain sections was: (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene > or = quinpirole > dopamine for putative dopamine agonists; spiperone > (+)-butaclamol > (-)-sulpiride > SCH 23390 >> cinanserin > (-)-butaclamol for antagonists. [3H]Quinpirole binding was decreased in the presence of guanine nucleotides in most brain regions except in the islands of Calleja and the molecular layer of cerebellar lobules 9 and 10. The regional distribution of [3H]quinpirole binding sites roughly paralleled the distribution of [3H]-(-)-sulpiride binding sites, with greatest densities present in the olfactory bulb glomerular layer, islands of Calleja, pituitary intermediate lobe, caudate/putamen, olfactory tubercles and nucleus accumbens. However, significantly greater densities of [3H]quinpirole binding than [3H]-(-)-sulpiride binding were observed in the molecular layer of cerebellar lobules 9 and 10, the islands of Calleja and olfactory bulb glomerular layer in concordance with the recently reported distribution of D3 receptor mRNA in these brain regions. Higher concentrations of [3H]quinpirole binding were also observed in the dorsomedial caudate and pituitary intermediate lobe. These data indicate the utility of [3H]quinpirole to label D3 as well as D2 dopamine receptors.

Immunohistochemical study of neurons containing corticotropin-releasing factor in Alzheimer's disease.

In the brains of controls and individuals with Alzheimer's disease (AD), antisera to corticotropin-releasing factor (CRF) were used to immunostain neurons and their processes. In AD, we identified abnormal CRF-immunoreactive axons as well as neurites associated with deposits of amyloid in brain regions showing senile plaques. The number of immunoreactive fibers was decreased in individuals with AD. In contrast, CRF immunoreactivity was markedly increased in some neurons located within the paraventricular nucleus (PVN) of the hypothalamus. These findings support previous neurochemical studies indicating that certain CRF systems are affected in AD.