Cytokine/neurotrophin interaction in the aged central nervous system.

Age-associated neurodegenerative diseases such as Alzheimer's disease are characterised by neuronal impairment that leads to cognitive deficits. As certain affected neurons depend on trophic factors such as neurotrophins (NTs), impairment in NT function has been suggested to be a component of neuronal damage associated with such disorders. Age-related neurodegenerative diseases are also characterised by high levels of proinflammatory cytokines such as tumour necrosis factor alpha (TNFalpha) in the CNS. Because TNFalpha receptors and certain NT receptors share a high degree of homology and are capable of activating similar signalling pathways, one possibility is that altered cytokine levels may affect NT function in the aged or diseased CNS. Here we wish briefly to review the evidence suggesting a role for cytokine and NT in the onset of age-associated neurodegenerative diseases. We propose that cytokine/NT interactions may alter neuronal homeostasis, thus possibly contributing to some of the neuronal degeneration occurring during such age-associated CNS diseases.

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-alpha.

The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-alpha (mERalpha) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERalpha by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH3/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERalpha, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERalpha and elicits the same response. A total of eight antibodies to ERalpha recognize mERalpha, making it likely that the membrane and nuclear proteins are highly related. Immuno-identification techniques have also been used to identify mERalpha on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH3/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERalpha-specific antibodies. The characteristics of mERalpha and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family.

HIV decreases glutamate transport in SK-N-MC neuroblastoma cells.

Autopsy studies of patients with AIDS dementia have shown neuronal loss consistent with a neurotoxic component of this disease. In vitro studies suggest that viral products or cytokines from HIV-infected macrophages (Mphi) may modulate or directly mediate excitotoxic cell death of neurons. Mphi differentiated from peripheral mononuclear blood cultures were infected with HIV, and conditioned media (CM) were harvested from these cultures. Exposure of SK-N-MC (neuroblastoma) cells to CM from HIV-infected Mphi for 4, 24 or > or = 48 h resulted in a mean suppression of 12-34% of the glutamate transport Vmax with no appreciable change in transport Km. An astrocytoma tumor cell, U373MG, showed similar CM-mediated glutamate uptake suppression. Changes were evident in total and Na+-dependent glutamate uptake, with significantly more suppression of Na+-dependent uptake. Similar effects were seen with the nonmetabolizable transporter agonist D-aspartate, indicating that the effect was on transport and not metabolism. No suppression was seen with CM from uninfected Mphi or Mphi infected with heat-inactivated HIV. The magnitude of uptake suppression was not correlated with CM p24 values, and removal of CM virions by ultracentrifugation and immunoprecipitation did not alter the uptake-suppressive properties of infected Mphi CM. Uptake suppression was seen when Mphi were infected with Mphi-tropic strains HIV(SF162), HIV(JR-CSF), HIV(NFN-SX) and a Mphi-tropic patient isolate, but not the lymphotropic strain HIV(LAI). HIV-infected Mphi may produce substances which suppress neuronal and glial glutamate neurotransmitter uptake, resulting in higher extracellular glutamate levels and leading possibly to deficits in cell signaling and neurotoxicity.

The other estrogen receptor in the plasma membrane: implications for the actions of environmental estrogens.

Environmental or nutritional estrogenic toxicants are thought to mediate developmental and carcinogenic pathologies. Estrogen receptor (ER) measurements are currently used to predict hormonal responsiveness; therefore all ER subpopulations should be considered. We have been involved in the immunoidentification and characterization of membrane steroid receptors in several systems and have recently shown that binding of estradiol (E2) to a subpopulation of ERs (mER) residing in the plasma membrane of GH3 pituitary tumor cells mediates the rapid release of prolactin (PRL). Here we review these findings and present other important characterizations of these receptors such as trypsin and serum susceptibility, movement in the membrane, confocal localization to the membrane, binding to and function of impeded ligands, and immunoseparation of cells bearing mER. We plan to use this system as a model for both the physiological and pathological nongenomic effects of estrogens and estrogenic xenobiotics. Specifically, it should be useful as an in vitro assay system for the ability of estrogenic xenobiotics to cause rapid PRL release as an example of nongenomic estrogen effects.

Circulating hyaluronan levels in the rodent: effects of age and diet.

Circulating hyaluronan (HA) levels were investigated as a function of age and diet in Fischer 344 male rats. A biphasic pattern of age-related changes was observed in rats fed ad libitum a diet in which the protein source was soya/fish meal. HA levels in 3- to 6- and 22- to 29-mo-old rats were not statistically different. However, HA levels in 12- to 20-mo-old rats were 10-29% of the levels in younger or aged adults. HA levels were also measured in rats fed ad libitum a semisynthetic diet in which the protein source was hydrolyzed casein. Whereas the two colonies exhibited similar biphasic age-related changes, HA levels differed 4- to 20-fold at every age examined. Caloric restriction affected HA levels in 19-mo-old casein-fed rats; HA levels were 2.3 times higher than age-matched controls and were not statistically different from young or aged animals. Serum and plasma HA levels were identical in the same individuals at all ages tested. These data suggest that HA turnover and metabolism in the rat are affected by age, dietary composition, and caloric intake.

Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding.

GH3/B6 rat pituitary tumor cells exhibit rapid prolactin release (within 5 min) when treated with nanomolar amounts of estrogen. However, the putative protein mediator of this nongenomic action has not been described. Using antibodies directed against a peptide representing the hinge region of the intracellular estrogen receptor (iER), we have demonstrated that these cells contain a membrane ER (mER). We now report that confocal scanning laser microscopy of cells labeled live with the anti-peptide antibody further supports a membrane localization of ER. The monoclonal antibodies H226 and H222 and a polyclonal antibody, ER21, each recognizing a unique epitope on iER (NH2 terminal to the DNA-binding region, within the steroid binding region, and the NH2-terminal end, respectively), also immunohistochemically label membrane proteins of immuno-selected GH3/B6 cells. These cells also specifically bind a fluorescent estrogen-BSA conjugate. Coincubation of cells with anti-ER antibody and the fluorescent estrogen-BSA conjugate reveals that these labels colocalize on cells. These results suggest that mER may be structurally similar to iER.

Membrane estrogen receptor-enriched GH(3)/B6 cells have an enhanced non-genomic response to estrogen.

We immunoselected GH(3)/B6 cells for a membrane estrogen receptor (mER) using antibodies generated against the rat intracellular ER (iER). Immunocytochemistry with anti-ER antibodies revealed bright fluorescence distributed in patches over the surface of mER-enriched cells, while cells immuno-depleted for mER showed only low-level membrane immunofluorescence. Quantitation via digital image analysis confirmed that immunoenriched populations show increases in both stained cell numbers and intensity of staining. Short-term culturing with serum reversibly decreased the intensity of immunostaining in mER-enriched cells to immuno-depleted cell levels. The mER-enriched populations initially contained ∼85% immunopositive cells in defined medium, but when cultured continuously with serum gradually decline to ∼22% immunopositive cells by 10 weeks. Cells enriched for mER showed a significant increase in rapid (after 2 or 5 min) prolactin release when treated with 17ß-estradiol, while mER-depleted cells lacked this response. Immunoprecipitabie membrane proteins isolated from mER-enriched cells were 60,000, 74,000 and ∼ 200,000 MW, compared to an iER size of 67,000. Therefore, the presence and level of an mER that is antigenically related to iER is correlated with the ability of GH(3)/B6 cells to mediate a rapid action of estrogen.

Physiological color change in squid iridophores. I. Behavior, morphology and pharmacology in Lolliguncula brevis.

Cephalopods generally are thought to have only static iridophores, but this report provides qualitative and quantitative evidence for active control of certain iridescent cells in the dermis of the squid Lolliguncula brevis. In vivo observations indicate the expression of iridescence to be linked to agonistic or reproductive behavior. The neuromodulator acetylcholine (ACh) induced dramatic opitcla changes in active iridophores in vitro, whereas ACh had little effect on passive iridophores elsewhere in the mantle skin. Bath application of physiological concentrations of ACh (10(-7)M to 10(-6)M) to excised dermal skin layers transformed the active iridophores from a non-reflective diffuse blue to brightly iridescent colors, and this reaction was reversible and repeatable. The speed of change to iridescent in vitro corresponded well to the speed of changes in the living animal. Pharmacological results indicate the presence of muscarinic receptors in this system and that Ca++ is a mediator for the observed changes. Although ACh is present in physiological quantities in the dermal iridophore layer, it is possible that ACh release is not controlled directly by the nervous system because electrophysiological stimulation of major nerves in the periphery resulted in no iridescence in L. brevis; nor did silver staining or transmission electron microscopy reveal neuronal elements in the iridophore layer. Thus, active iridophores may be controlled by ACh acting as a hormone.