Effects of diet and obesity on brain α1- and α2-noradrenergic receptors in the rat

The chronic feeding of a sweetened condensed milk/corn oil diet (CM diet) to adult male rats produced significant increases in body weight and levels of plasma insulin in 34% of the rats fed this diet with respect to chow-fed controls. Levels of alpha 1-noradrenergic receptor binding were lower (32%) in the hypothalamic ventromedial nucleus (VMN) of only those rats which became obese (DIO rats) with respect to both chow-fed controls and those rats which resisted the development of obesity on the CM diet (DR rats). Also, alpha 1-noradrenergic binding was inversely proportional to body weight gain in the VMN (r = -0.831). alpha 2-Noradrenergic receptors were 30-37% lower in both the DIO and DR rats in the dorsomedial nucleus and dorsal area of the hypothalamus, and the medial dorsal area and nucleus reuniens of the thalamus. The similar decreases in alpha 2-noradrenergic receptors in both the DIO and DR rats in these areas suggested that dietary factors alone were responsible for these changes. There were no significant differences from chow-fed rats for hypothalamic dopamine (D2) or beta-noradrenergic (beta 1- and beta 2-) receptors in either DR or DIO rats. These results indicate that VMN alpha 1-noradrenergic receptors co-vary with body weight and implicate a role for alpha 1-receptors in the VMN in the central neuronal regulation of body weight.     

α1-Adrenergic receptors and stimulation of [H]inositol metabolism in rat brain: Regional distribution and parallel inactivation

The relationship between norepinephrine-stimulated phosphatidyl-inositol metabolism andα₁-adrenergic receptor density was examined in rat brain. Increases in phosphatidyl-inositol metabolism were determined by accumulation of [³H]inositol phosphates in the presence of lithium in brain slices, while receptor density was determined by specific binding of¹²⁵I-BE 2254 (¹²⁵IBE) in membrane fractions. Treatment of slices of cerebral cortex with increasing concentrations of the irreversibleα₁-adrenergic receptor antagonist phenoxybenzamine caused a parallel inactivation of specific¹²⁵IBE binding sites and norepinephrine-induced [³H]inositol phosphate accumulation, although approximately 20% of the binding sites remained after abolition of the inositol response. Comparison of the density of¹²⁵IBE binding sites and the magnitude of norepinephrine-stimulated [³H]inositol phosphate accumulation in 8 different brain regions did not show a particularly good correlation. The thalamus had the highest density of binding sites and an intermediate inositol response, while the hippocampus had the highest inositol response but an intermediate density of binding sites. However, the cerebellum had the lowest density of binding sites and no measurable inositol response. Treatment of slices of each region with 300 nM phenoxybenzamine abolished the inositol response and caused a 59–73% decrease in the density of¹²⁵IBE binding sites. The lack of correlation between receptor density and inositol response between brain regions could not be explained on the basis of receptor affinity, spare receptors, protein content, nor differences in slice size. These results suggest thatα₁-adrenergic receptors labeled by¹²⁵IBE are coupled to [³H]inositol metabolism in rat brain, but the receptor density is not the sole determinant of the magnitude of norepinephrine-induced increases in [^3H]inositol metabolism.     

Tritium-sensitive film autoradiography of guinea pig brain α2-noradrenergic receptors

Tritium-sensitive film autoradiography was used to determine the distribution of α-noradrenergic receptors (i.e. [³H] p-aminoclonidine binding sites) in guinea pig forebrain. α₂-Receptors are heterogeneously distributed throughout the forebrain. Many limbic system structures, such as bed nucleus of stria terminalis, medial preoptic area, medial amygdaloid nucleus and lacunosum molecular layer in hippocampus were heavily labeled. We did not quantify receptor density in areas containing principally white matter but the optical density in those areas was similar to film background suggesting a very low receptor density. Low receptor concentrations were also found in areas that do not contain a high percentage of white matter, such as lateral septum and ventromedial hypothalamic nucleus.     

Autoradiographic studies of central α2A- and α2C-adrenoceptors in the rat using [H]MK912 and subtype-selective drugs

In the present study we examined the distribution of α_2A- and α_2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between α_2A- and α_2C-adrenoceptors, the slices were incubated with [³H]MK912 in the presence of graded concentrations of the α_2A-selective drug, BRL44408, or the α_2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [³H]MK912 plus 185 nM BRL44408 selectively labeled α_2C-adrenoceptors, while 0.4 nM [³H]MK912 plus 220 nM spiroxatrine selectively labeled α_2A-adrenoceptors. Using this approach, α_2C-adrenoceptors were detected in the striatum, while α_2A-adrenoceptors predominated in the cortical layers 1–4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.     

Subtypes of β-adrenergic receptors in rat cerebral microvessels

The¹²⁵I-labeled iodohydroxybenzylpindolol (IHYP) binding to β-receptors on brain microvessels is inhibited by isoproterenol, epinephrine and norepinephrine, with K_i values of 2 × 10⁻⁷M, 2.5 × 10⁻⁶M and 1.2 × 10⁻⁵M, respectively. A modified Scatchard analysis of the inhibitory effects of practolol, metroprolol and zinterol on IHYP binding has shown that the proportion of β₂-receptors in our preparation is about 80% of the total β-adrenergic receptor population. Our data indicate that the β-adrenergic receptors located on cerebral microvessels are of both β₁ and β₂ types, with a predominance of the β₂ type.     

Progesterone, 5α-pregnane-3,20-dione and 3α-hydroxy-5α-pregnane-20-one in specific regions of the human female brain in different endocrine states

Post-mortem concentrations of progesterone, 5α-pregnane-3,20-dione (5α-DHP) and 3α-hydroxy-5α-pregnane-20-one (allopregnanolone) were measured in 17 brain areas and serum in five fertile and five postmenopausal women. Steroid concentrations were measured with radioimmunoassay after extraction of brain tissue with ethanol and purification with celite chromatography. There were regional differences in brain concentrations of all three steroids. The highest progesterone levels were noted in the amygdala, cerebellum and hypothalamus and the highest levels of 5α-DHP and allopregnanolone were seen in the substantia nigra and basal hypothalamus. Brain concentrations of all three steroids were significantly higher in the fertile women in luteal phase compared to their postmenopausal controls (P<0.01). In general, the study showed that there is a variation in brain concentrations depending on ovarian steroid production, indicating that the secretion pattern during the menstrual cycle is reflected in the brain. However, regional differences in brain steroid levels imply local mechanisms for steroid uptake and binding as well. Investigations of gonadal steroid distributions in the human brain might be of importance considering the actions of these steroids in the central nervous system. Such studies could provide information about physiological mechanisms, such as the ovulation, and also form a baseline for comparative studies of normal and pathological conditions involving steroids, for instance, catamenial epilepsy and the premenstrual tension syndrome.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Synthesis, metabolism and levels of the neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), in rat pituitaries

The neuroactive steroid, 3α-hydroxy-4-pregnen-20-one (3αHP), is a metabolite of progesterone and a precursor of 3α-hydroxy-5α-pregnan-20-one (5αP3α; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA_A receptor complex, 3αHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca²⁺-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3αHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3αHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with ³H- or ¹⁴C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3αHP along with 5αP3α, 5α-pregnane-3,20-dione, 20α-hydroxy-5α-pregnan-3-one, 3β-hydroxy-5α-pregnan-20-one, 5α-pregnane-3α(β), 20α-diols, 20α-hydroxy-4-pregnen-3-one, and 4-pregnene-3α(β), 20α-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3α-hydroxysteroid oxidoreductase (3α-HSO), 20α-HSO, 3β-HSO, and 5α-reductase. The activities of 5α-reductase and 3α-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3αHP had been formed. The accumulation of 3αHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7-³H]3αHP showed that pituitary cells convert about 29% and 8% of the 3αHP to progesterone and 5αP3α, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3αHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3αHP were about 2–3-fold greater than those of progesterone in the same pituitaries. In older (80–100 days old) cycling rats, the levels of 3αHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3αHP, from progesterone, and in turn to metabolize 3αHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3αHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3αHP may be (in part, or in whole) via paracrine or autocrine mechanisms.     

Behavior and binding: Correlations between α1-adrenergic stimulation of acoustic startle and α1-adrenoceptor occupancy and number in rat lumbar spinal cord

The relationship between alterations in α₁-adrenoceptors and behavioral effects of α₁-adrenergic agonists were investigated in a localized region of the rat central nervous system. Direct infusion of the α₁-adrenergic agonists,d-amphetamine or phenylephrine. into the subarachnoid space of the lumbar cord (intrathecal administration) increased the amplitude of the acoustic startle reflex, The magnitude of this behavioral facilitation correlated highly with the degree of α₁-adrenoceptor occupation measured by [³H]prazosin binding in lumbar spinal tissue. Using an in vitro estimate of receptor occupation, maximal potentiation of startle occurred following approximately 30% occupation of the receptors, using eitherd-amphetamine or phenylephrine. Intrathecal administration of 6-OHDA produced a 95% decrease in spinal norepinephrine and markedly enhanced the behavioral response to intrathecal phenylephrine as well as the number of α₁-adrenoceptors. The correlation between the time course of the behavioral and binding changes was 0.99. No change in receptor affinity (K_D) or receptor occupation by phenylephrine was found after 6-OHDA. The data indicate that receptor binding parameters do have predictive value for behavior, especially if localized regions of the nervous system, critical to the behavior, are analyzed.     

The anti-amnesic effects of sigma1 (σ1) receptor agonists confirmed by in vivo antisense strategy in the mouse

The sigma₁ (σ₁) receptor cDNA was recently cloned in several animal species, including the mouse. In order to firmly establish the implication of σ₁ receptors in memory, a phosphorothioate-modified antisense oligodeoxynucleotide (aODN) targeting the σ₁ receptor mRNA and a mismatched analog (mODN) were administered intracerebroventricularly for 3 days in mice. Scatchard analyses of in vitro (+)-[³H]SKF-10,047 binding to σ₁ sites showed that B_max values were significantly decreased in the hippocampus (−58.5%) and cortex (−38.1%), but not in the cerebellum, of aODN treated mice, as compared to saline- or mODN-treated animals. In vivo binding levels were also significantly decreased after aODN treatment in the hippocampus and cortex but not in the cerebellum. The anti-amnesic effects of the selective σ₁ agonists PRE-084 or SA4503 were evaluated against the learning impairments induced by dizocilpine or scopolamine, respectively, using spontaneous alternation behavior and passive avoidance task. The anti-amnesic effects of PRE-084 or SA4503, observed after saline- or mODN-treatment, were blocked after aODN administration. These observations bring a molecular basis to the modulatory role of σ₁ receptors in memory processes.     

Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (α4β2) transfected into mouse fibroblasts with ion channel activity

(−)-Nicotine, cytisine and carbachol evoked⁸⁶Rb efflux from mouse fibroblasts stably transfected with α₄β₂ chick brain nicotinic subunits. This response to (−)-nicotine was inhibited by mecamylamine and dihydro-β-erythroidine and was mirrored by a rise in intracellular Ca²⁺ measured by microspectrofluorimetry. Lobeline and isoarecolone methiodide evoked no significant⁸⁶Rb from cells and unlike the above agonists displayed significantly different IC₅₀ values for the displacement of [³H]nicotine from mammalian (rat and human cerebral cortex) and transfected fibroblast membranes.     

Reduced antiheparin effect of desialyzed α1-acid glycoprotein

α₁-Acid glycoprotein (α₁-acid GP) of human plasma counteracts the heparin-accelerated, antithrombin III-mediated inactivation of thrombin and factor Xa. In some preparations of α₁-acid GP which had a reduced antiheparin effect, less than normal amounts of sialic acid were found. No antiheparin activity remained after almost complete desialization of the purified glycoprotein with insolubilized neuraminidase. The significance of the sialyl residues for the antiheparin effect of α₁-acid GP is discussed.     

Estradiol modulation of α2-noradrenergic receptors in guinea pig brain assessed by tritium-sensitive film autoradiography

In this experiment, we examined the influence of estradiol on α₂-noradrenergic receptor binding in the female guinea pig brain with tritium-sensitive film autoradiography. Treatment of ovariectomized guinea pigs with estradiol increased α₂-receptor binding in regions of the preoptic area and decreased binding in the ventromedial nucleus of the hypothalamus. There was no effect of estradiol on α₂-receptor concentrations in other estradiol concentrating regions or in brain areas which do not concentrate estradiol.     

Cerebral vessels express interleukin 1β after focal cerebral ischemia

Rapid and marked increased levels of expression of interleukin 1β (IL-1β) mRNA have been detected in animal models of cerebral ischemia. However, the protein production of IL-1β and the cellular sources of IL-1β are largely undefined after cerebral ischemia. In the present study, we have measured the cellular localization of IL-1β protein in brain tissue from non-ischemic and ischemic mice using immunohistochemistry. Male C57B/6J (n=45) mice were subjected to middle cerebral artery (MCA) occlusion by a clot or a suture. The mice were sacrificed at time points spanning the period from 15 min to 24 h after onset of the MCA occlusion. Non-operated and sham-operated mice were used as control groups. A monoclonal anti-IL-1β antibody was used to detect IL-1β. In the non-operated and sham-operated mice, a few IL-1β immunoreactive cells were detected scattered throughout both hemispheres. IL-1β immunoreactive cells increased in the ischemic lesion as early as 15 min and peaked at 1 h to 2 h after MCA occlusion. IL-1β immunoreactivity was detected in the cortex of the contralateral hemisphere 1 h after ischemia. By 24 h after onset of ischemia, IL-1β immunoreactivity was mainly present adjacent to the ischemic lesion and in the non-ischemic cortex. IL-1β immunoreactivity was found on endothelial cells and microglia. This study demonstrates an early bilateral expression of IL-1β on endothelium after MCA occlusion in mice.     

Recombinant variant fibrinogens substituted at residues γ326Cys and γ339Cys demonstrated markedly impaired secretion of assembled fibrinogen

Background

To study the functions of residues γ326Cys and γ339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring γ326Tyr and γ326Ser variants, along with γ326Ala and γ339Ala variants.

Methods

A fibrinogen γ-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed.

Results

The CHO cells synthesized mutant γ-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen.

Conclusions

The present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the γ-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of γ326Tyr and γ326Ser suggest that heterozygous fibrinogen molecules containing variant γ-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia.     

Regulation of central α-adrenoceptors by serotoninergic denervation

α₁- and α₂-adrenoceptors were assessed by binding studies using [³H]prazosin and [³H]p-aminoclonidine as ligands in membrane preparations from the cortex, hippocampus and hypothalamus of rats, 3 weeks after intracerebroventricular injection of the neurotoxin 5,7-dihydroxytryptamine. Cortical α₁ and hippocampal α₂ adrenoceptors were significantly increased. Treatment also affected the affinity of cortical α₂ adrenoceptors. These results suggest a heterologous, region-specific regulation of both subtypes of central α-adrenergic receptors by serotonin.