Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia.

Activated microglia surrounding amyloid beta-containing senile plaques synthesize interleukin-1, an inflammatory cytokine that has been postulated to contribute to Alzheimer's disease pathology. Studies have demonstrated that amyloid beta treatment causes increased cytokine release in microglia and related cell cultures. The present work evaluates the specificity of this cellular response by comparing the effects of amyloid beta to that of amylin, another amyloidotic peptide. Both lipopolysaccharide-treated THP-1 monocytes and mouse microglia showed significant increases in mature interleukin-1beta release 48 h following amyloid beta or human amylin treatment, whereas nonfibrillar rat amylin had no effect on interleukin-1beta production by THP-1 cells. Lipopolysaccharide-stimulated THP-1 cells treated with amyloid beta or amylin also showed increased release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokines interleukin-8 and macrophage inflammatory protein-1alpha and -1beta. THP-1 cells incubated with fibrillar amyloid beta or amylin in the absence of lipopolysaccharide also showed significant increases of both interleukin-1beta and tumor necrosis factor-alpha mRNA. Furthermore, treatment of THP-1 cells with amyloid fibrils resulted in an elevated expression of the immediate-early genes c-fos and junB. These studies provide further evidence that fibrillar amyloid peptides can induce signal transduction pathways that initiate an inflammatory response that is likely to contribute to Alzheimer's disease pathology.

Characterization of multiple forms of the human glycine transporter type-2.

The human glycine transporter type 2 (hGlyT2) was cloned from a spinal cord cDNA library using PCR-based methodologies. The isolated sequence exhibits 89% homology with the previously isolated rat GlyT2 cDNA (Liu et al., J. Biol. Chem. 268 (1993) 22802-22808) at the nucleotide level, and 93% amino acid sequence identity. The greatest divergence between the human and rat sequences is found at the amino-terminus, where only 74% amino acid identity exists in residues 1-190. Expression of the intact hGlyT2 transporter sequence in COS-7 cells resulted in a 10-fold increase in high-affinity uptake relative to control cells transfected with vector alone. An artificially truncated form of the transporter, missing the NH2-terminal 153 amino acids, was also capable of mediating glycine uptake. However, an identified variant lacking the first 234 amino acids was non-functional. An hGlyT2 transporter containing a 14-residue deletion in the intracellular loop between transmembrane domains 6 and 7 was also identified and expressed, but failed to mediate glycine uptake. Like rat GlyT2, the high-affinity uptake mediated by hGlyT2 was found to be insensitive to the GlyT1 inhibitor sarcosine.

D2S, D2L, D3, and D4 dopamine receptors couple to a voltage-dependent potassium current in N18TG2 x mesencephalon hybrid cell (MES-23.5) via distinct G proteins.

We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.

D4 dopamine receptor binding affinity does not distinguish between typical and atypical antipsychotic drugs.

The affinities of 13 atypical and 12 typical antipsychotic drugs for the cloned rat D4 dopamine receptor and the D4/D2 ratios were examined. Of the atypical antipsychotic drugs tested, only clozapine, risperidone, olanzapine, zotepine and tiospirone had affinities less than 20 nM. In fact, many atypical antipsychotic drugs had relatively low affinities for the cloned rat D4 receptor, with Ki values greater than 100 nM (Seroquel, fluperlapine, tenilapine, FG5803 and melperone). Additionally, several typical antipsychotic drugs had high affinities for the cloned rat D4 receptor, with Kis less than 20 nM (loxapine, chlorpromazine, fluphenazine, mesoridazine, thioridazine and trifluoroperazine). The ratios of D2/D4 affinities did not differentiate between these two types of antipsychotic drugs. Thus, D4 dopamine receptor affinity, used as a single measure, does not distinguish between the group of typical and atypical antipsychotic drugs analyzed.

Estrogen receptor mRNA alterations in the developing rat hippocampus.

We previously reported transiently elevated ER protein levels in the postnatal rat hippocampus suggesting that this brain region may be sensitive to estrogenic trophic and organizational influences during a 'critical period' of sexual differentiation. In order to examine whether alterations in ER gene expression underlie the ontogenetic pattern of the hippocampal ER, we examined ER mRNA levels over the early postnatal period and in adult rats. This was accomplished by both a highly quantitative RNase protection assay and in situ hybridization histochemistry. Hippocampal ER mRNA levels increased significantly (P < 0.005) between birth and postnatal day (PDN) 4 when peak concentrations were found and then declined by PND-10. Adult male hippocampal ER mRNA values were similar to those found in newborn and PND-10 animals but were significantly less (P < 0.05) than those observed on PND-4. Results from the in situ hybridization experiments correlated well with those from the RNase protection analysis. High levels of ER mRNA were present in the CA3 pyramidal layer with somewhat lower labeling intensities present in CA1 and the dentate gyrus of the PND-4 animal. In contrast, adult male animals demonstrated little hybridization throughout the hippocampus. Thus, the temporal pattern in ER mRNA levels in the hippocampus found in the present study correlates well with our previous developmental profile of the ER protein. These findings suggest that the ontogeny of ER in the hippocampus is regulated by alterations in ER gene expression in specific neuronal populations.

Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis.

The rapid activation of stress-responsive neuroendocrine systems is a basic reaction of animals to perturbations in their environment. One well-established response is that of the hypothalamo-pituitary-adrenal (HPA) axis. In rats, corticosterone is the major adrenal steroid secreted and is released in direct response to adrenocorticotropin (ACTH) secreted from the anterior pituitary gland. ACTH in turn is regulated by the hypothalamic factor, corticotropin-releasing hormone. A sex difference exists in the response of the HPA axis to stress, with females reacting more robustly than males. It has been demonstrated that in both sexes, products of the HPA axis inhibit reproductive function. Conversely, the sex differences in HPA function are in part due to differences in the circulating gonadal steroid hormone milieu. It appears that testosterone can act to inhibit HPA function, whereas estrogen can enhance HPA function. One mechanism by which androgens and estrogens modulate stress responses is through the binding to their cognate receptors in the central nervous system. The distribution and regulation of androgen and estrogen receptors within the CNS suggest possible sites and mechanisms by which gonadal steroid hormones can influence stress responses. In the case of androgens, data suggest that the control of the hypothalamic paraventricular nucleus is mediated trans-synaptically. For estrogen, modulation of the HPA axis may be due to changes in glucocorticoid receptor-mediated negative feedback mechanisms. The results of a variety of studies suggest that gonadal steroid hormones, particularly testosterone, modulate HPA activity in an attempt to prevent the deleterious effects of HPA activation on reproductive function.

Neuroendocrine and neurochemical responses to novelty stress in young and old male F344 rats: effects of d-fenfluramine treatment.

To understand some of the mechanisms underlying the neuroendocrine and neurochemical changes associated with aging, we administered the serotonin [5-hydroxytryptamine (5-HT)] releaser and reuptake inhibitor d-fenfluramine (d-FEN; 0.0, 0.2, or 0.6 mg/kg/day, p.o) for 30-38 days to young (4 months) and old (22 months) F344 male rats. Rats were stressed by placement into a novel open field (OF) for 20 min before sacrifice. Control animals were sacrificed immediately upon removal from their home cage (HC). Old rats exhibited less (p < 0.05) exploratory behavior than young rats, which was not altered by treatment with d-FEN. Old HC rats also had higher (p < 0.05) basal plasma levels of adrenocorticotropic hormone (ACTH) and prolactin (PRL) than young HC rats. Old OF rats showed higher (p < 0.05) levels of ACTH and corticosterone (CORT) than young OF animals. A stress-induced increase in PRL secretion was not observed in old rats. Subchronic low-dose d-FEN normalized the enhanced ACTH and CORT responses of old animals to novelty. In addition to these endocrine changes, stress-induced increases in medial frontal cortex (MFC) dopamine (DA) and norepinephrine (NE) turnover also were observed. The increase in NE turnover was greater (p < 0.01) in old than in young rats. d-FEN treatment blocked the stress-induced increase in MFC NE but not MFC DA turnover in both young and old rats. These data support a role for 5-HT and/or NE in some age-related neuroendocrine perturbations and suggest that increased 5-HT neurotransmission can normalize the hyperactivation of the hypothalamo-pituitary-adrenal axis of old male rats.

Hormonal regulation of androgen receptor mRNA in the brain and anterior pituitary gland of the male rat.

To determine possible cellular mechanisms governing androgen action in the brain, we examined the hormonal regulation of androgen receptor (AR) mRNA in neural tissues by Northern blot hybridization and RNase protection analysis. While a single hybridizable species of AR mRNA of approximately 11 kb was found in the anterior pituitary gland (AP) and ventral prostate gland (VP), an additional species of AR mRNA, approximately 2 kb smaller, was revealed in neural tissues. Furthermore, in these neural tissues, hormonal regulation of the two species of mRNA was coordinated; long-term castration increased levels of both forms, while testosterone replacement reduced them. The same pattern of regulation was observed for the single 11 kb form in the AP. An RNase protection assay was validated and utilized to quantitatively analyze the hormonal regulation of AR mRNA. Castration (4 days) resulted in significantly increased AR mRNA in the AP and hypothalamic-preoptic area, but not the amygdala, which subsequent administration of dihydrotestosterone (DHT; 1 day; 2 mg/animal) significantly decreased. In the AP, administration of estradiol benzoate (EB) for 1 or 5 days also reversed this effect. However, EB treatment increased the amount of total RNA isolated per gland. Consequently, when the data are normalized to RNA content per gland, 5 days of EB treatment resulted in a significant increase in AR mRNA content. These findings suggest that in contrast to the AP and VP, two forms of androgen receptor mRNA exist in the brain. In addition, there appears to be tissue and hormone specific regulation of AR mRNA.

Estrogen-induced alterations in the regulation of mineralocorticoid and glucocorticoid receptor messenger RNA expression in the female rat anterior pituitary gland and brain.

The influence of estrogen (E) on corticosterone (CORT) receptor function in neural tissue was investigated in female Sprague-Dawley rats. This was accomplished by using a sensitive solution-hybridization RNase protection assay to examine the effect of E on the regulation of CORT receptor mRNAs. Animals were bilaterally ovariectomized (OVX), and a Silastic capsule (0.5 cm) containing 17beta-estradiol was sc implanted. Control animals received a blank capsule. Animals were killed 1, 7, or 21 days later. Anterior pituitary glucocorticoid receptor (GR) mRNA levels were significantly lower (P < 0.01) in E-treated rats at all time points examined. Hippocampal GR mRNA levels were significantly decreased below OVX values (P < 0.01) after 1 and 21 days of E treatment. Hypothalamic-preoptic area (HPOA) GR mRNA levels were significantly lower (P < 0.01) than OVX values only after 21 days of E treatment. Mineralocorticoid receptor mRNA levels were significantly lower after E treatment (P < 0.01) at all time points and in all three tissues examined. In a second study, we administered the GR-specific agonist RU 28362 (40 mug/100 g BW for 4 days) or the nonspecific agonist dexamethasone (DEX; 40 mug/100 g BW for 4 days) to OVX - and OVX + E-treated animals. The administration of RU 28362 significantly down-regulated hippocampal GR mRNA (P < 0.05) in OVX rats only. In contrast, DEX administration significantly down-regulated hippocampal GR mRNA (P < 0.05) in both control and E-treated animals. The administration of DEX or RU 28362 significantly reduced GR mRNA levels (P < 0.05) in the HPOA of OVX control animals, but not E-treated animals. Thus, E treatment results in a loss of the glucocorticoid receptor's ability to down-regulate its mRNA. These studies, combined with our earlier findings that E treatment impairs the ability of GR to down-regulate its protein (8), provide evidence that E interferes with glucocorticoid receptor function.

Chronic estrogen-induced alterations in adrenocorticotropin and corticosterone secretion, and glucocorticoid receptor-mediated functions in female rats.

The effect of estrogen (E) on the hypothalamic-pituitary-adrenal axis was investigated in female Sprague-Dawley rats. Animals were bilaterally ovariectomized (OVX), and a Silastic capsule (0.5 cm) containing 17 beta-estradiol was sc implanted. Control animals received a blank capsule. Animals were killed 21 days later. In E-treated rats, we found significantly higher corticosterone (CORT) peak levels 20 min after a 5-sec footshock (1.0 mamp) or exposure to ether vapors (P less than 0.05) compared to those in OVX controls. In addition, the recovery of the ACTH and CORT responses to footshock stress was significantly prolonged (P less than 0.05) in the presence of E. Furthermore, the ACTH and CORT secretory responses to ether stress could be suppressed by exogenous RU 28362 (a specific glucocorticoid receptor agonist; 40 micrograms/100 g BW for 4 days) in OVX controls (P less than 0.05), but not in E-treated animals. These data suggest that E can impair glucocorticoid receptor-mediated delayed or slow negative feedback. Consequently, we examined the influence of E on mineralocorticoid and glucocorticoid receptor concentrations using in vitro binding assays. E did not alter mineralocorticoid or glucocorticoid receptor concentrations in any of the brain regions examined. The administration of RU 28362 (40 micrograms/100 g BW for 4 days) to OVX control or E-treated rats significantly down-regulated hippocampal glucocorticoid receptor (P less than 0.02) in control rats only. In contrast, aldosterone administration (40 micrograms/100 g BW for 4 days) significantly down-regulated hippocampal glucocorticoid receptor (P less than 0.0008) in both control and E-treated animals. Thus, E treatment results in a loss of the glucocorticoid receptor's ability to autoregulate; this suggests that E may cause a functional impairment of the glucocorticoid receptor even though receptor binding appears normal. These findings suggest that hyperactivation of the hypothalamic-pituitary-adrenal axis after stress in E-treated rats is due in part to impaired glucocorticoid receptor-mediated slow negative feedback.