Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.

INTRODUCTION: We examined the participation of a membrane form of estrogen receptor (mER)-alpha in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells. METHODS: We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-alpha-enriched (mERhigh) and mER-alpha-depleted (mERlow) populations. We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha. Western analysis was used to determine colocalized estrogen receptor (ER)-alpha and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory. RESULTS: Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells, whereas the majority of mERlow cells exhibited little or no staining. Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2, and that ER-alpha was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A. CONCLUSION: Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E2-induced cell proliferation outcomes in these cell types.

Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses.

INTRODUCTION: 17beta-estradiol (E2) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mERlow) and high (mERhigh) expression of the membrane form of estrogen receptor (mER)-alpha, and thus addressed the receptor subform involved in cAMP signaling. METHODS: MCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mERhigh (mER-alpha-enriched) and mERlow (mER-alpha-depleted) populations. Unique (compared with previously reported) incubation conditions at 4 degrees C were found to be optimal for demonstrating E2-induced cAMP production. Time-dependent and dose-dependent effects of E2 on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E2-induced cell proliferation were assessed using the crystal violet assay. RESULTS: We demonstrated a rapid and transient cAMP increase after 1 pmol/l E2 stimulation in mERhigh cells; at 4 degrees C these responses were much more reliable and robust than at 37 degrees C (the condition most often used). The loss of cAMP at 37 degrees C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mERhigh cells than in mERlow cells, implicating mER-alpha levels in the process. ICI172,780 blocked the E2-induced response and 17alpha-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E2 dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E2 concentrations in mERhigh cells. Proliferation of mERlow cells was stimulated over the whole range of E2concentrations, whereas the number of mERhigh cells was greatly decreased at concentrations above 1 nmol/l, suggesting that estrogen over-stimulation can lead to cell death, as has previously been reported, and that mER-alpha participates. E2-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses. CONCLUSION: Rapid mER-alpha-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number.

Some components of hibernation rhythms.

At low ambient temperatures some small mammals drastically reduce their body temperature and enter a state of dormancy known as hibernation. They exhibit endogenous rhythms of heterothermy/homeothermy and body mass fluctuations with the period close to one year. At high ambient temperature only body mass cycling is expressed for almost one year as well. The effect of temperature on circannual periods of some biological rhythms in different hibernators in free-running conditions as well as the circaseptal character of the intrahibernation rhythm of periodic arousals are reviewed.

Radoslav K. Andjus (1926-2003): a brief summary of his life and work.

Radoslav K. Andjus was a professor of physiology and biophysics at the University of Belgrade, Serbia, from 1953 to 1992. He was an elected member of the Serbian Academy of Sciences and Arts, the International Academy of Astronautics, and the Montenegrin Academy of Sciences and Arts. He published over 190 papers in domestic and international journals and three textbooks. The main field of his research was thermophysiology. He studied hypothermia, suspended animation and resuscitation, hibernation and biological rhythms, temperature adaptation and acclimation, and cryoprotection. Professor Andjus also contributed significantly to the fields of brain metabolism, endocrinology, electroretinography, as well as biophysical modeling and theoretical biology.

Mechanisms of immediate temperature compensation: experiments with brain synaptosomes from rat and ground squirrel.

Glucose conversion by brain synaptosomes can be regarded as a special case of intrinsic kinetic properties of the enzyme substrate system. Temperature modulation of apparent K(m) for this process can be described with our kinetic model. Using experimental data and the kinetic model, the minimal K(m) value for glucose conversion in ground squirrel synaptosomes was found at the lower temperature (6.5 degrees C), much lower than that for the rat (16.6 degrees C). The inversion temperatures (T(min)) closely coincided with the lowest body temperatures from which the unassisted recovery from hypothermia was demonstrated in both species. This study indicated that thermal modulation of enzyme affinities may have an adaptive role in endotherms that is linked to their tolerance to hypothermia.

Cell-type specific messenger functions of extracellular calcium in the anterior pituitary.

Calcium can serve not only as an intracellular messenger, but also as an extracellular messenger controlling the gating properties of plasma membrane channels and acting as an agonist for G protein-coupled Ca(2+)-sensing receptors. Here we studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of the extracellular Ca(2+) concentration ([Ca(2+)]e) induced transient elevations in the intracellular Ca(2+) concentration ([Ca(2+)]i), and elevations in [Ca(2+)]e above physiological levels decreased [Ca(2+)]i in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of [Ca(2+)]i responses depended on the [Ca(2+)]e and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated Ca(2+) influx. Changes in [Ca(2+)]e also affected GH and PRL secretion. The PRL secretory profiles paralleled the [Ca(2+)]i profiles in lactotrophs, whereas GH secretion was also stimulated by [Ca(2+)]e independently of the status of voltage-gated Ca(2+) influx. [Ca(2+)]e modulated GH secretion in a dose-dependent manner, with EC(50) values of 0.75 and 2.25 mM and minimum secretion at about 1.5 mM. In a parallel experiment, cAMP accumulation progressively increased with elevation of [Ca(2+)]e, whereas inositol phosphate levels were not affected. These results indicate the cell type-specific role of [Ca(2+)]e in the control of Ca(2+) signaling and secretion.

The cancer drug tamoxifen: a potential therapeutic treatment for spinal cord injury.

Tamoxifen (TMX) is a selective estrogen receptor modulator that can mimic the neuroprotective effects of estrogen but lacks its systemic adverse effects. We found that TMX (1 mg/day) significantly improved the motor recovery of partially paralyzed hind limbs of male adult rats with thoracic spinal cord injury (SCI), thus indicating a translational potential for this cancer medication given its clinical safety and applicability and the lack of currently available treatments for SCI. To shed light on the mechanisms underlying the beneficial effects of TMX for SCI, we used proteomic analyses, Western blots and histological assays, which showed that TMX treatment spared mature oligodendrocytes/increased myelin levels and altered reactive astrocytes, including the upregulation of the water channels aquaporin 4 (AQP4), a novel finding. AQP4 increases in TMX-treated SCI rats were associated with smaller fluid-filled cavities with borders consisting of densely packed AQP4-expressing astrocytes that closely resemble the organization of normal glia limitans externa (in contrast to large cavities in control SCI rats that lacked glia limitans-like borders and contained reactive glial cells). Based on our findings, we propose that TMX is a promising candidate for the therapeutic treatment of SCI and a possible intervention for other neuropathological conditions associated with demyelination and AQP4 dysfunction.

Inhibition of IL-6 signaling: A novel therapeutic approach to treating spinal cord injury pain.

To characterize the contribution of interleukin-6 (IL-6) to spinal cord injury pain (SCIP), we employed a clinically relevant rat contusion model of SCIP. Using Western blots, we measured IL-6 levels in lumbar segments (L1-L5), at the lesion site (T10), and in the corresponding lumbar and thoracic dorsal root ganglia (DRG) in 2 groups of similarly injured rats: (a) SCI rats that developed hind-limb mechanical allodynia (SCIP), and (b) SCI rats that did not develop SCIP. Only in SCIP rats did we find significantly increased IL-6 levels. Immunocytochemistry showed elevated IL-6 predominantly in reactive astrocytes. Our data also showed that increased production of IL-6 in hyperreactive astrocytes in SCIP rats may explain still-poorly understood astrocytic contribution to SCIP. To test the hypothesis that IL-6 contributes to mechanical allodynia, we treated SCIP rats with neutralizing IL-6 receptor antibody (IL-6-R Ab), and found that one systemic injection abolished allodynia and associated weight loss; in contrast to gabapentin, the analgesic effect lasted for at least 2weeks after the injection, despite the shorter presence of the Ab in the circulation. We also showed that IL-6-R Ab partially reversed SCI-induced decreases in the protein levels of the glutamate transporter GLT-1 12hours and 8days after Ab injection, which may explain the lasting analgesic effect of the Ab in SCIP rats. A link between reactive astrocytes IL-6-GLT-1 has not been previously shown. Given that the humanized IL-6-R Ab tocilizumab is Food and Drug Administration-approved for rheumatoid arthritis, we are proposing tocilizumab as a novel and potentially effective treatment for SCIP.

Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits.

The G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the G i/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to G i/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity.

Kinetic properties of the enzyme-substrate system: a basis for immediate temperature compensation.

One-minimum U-shaped temperature profiles of the dissociation constant (K(m)) have been observed experimentally with a variety of enzyme-substrate (E-S) systems. The increase of E-S affinity with falling temperature ("positive thermal modulation of affinity"), which opposes the cold-induced reduction in catalytic velocity, has been often interpreted as significant for both immediate and evolutionary temperature compensations and of major importance in setting thermal limits in ectothermic organisms. This role was denied to enzymes from endotherms, on the ground that their minimal K(m) values were situated well below their normal body temperature. Evidence is presented in this report that affinity changes described by U-shaped profiles can simply be the consequence of intrinsic kinetic properties of the E-S system. Theoretical modeling is achieved by combining the classical expression for the Michaelis constant with Transition State Theory expressions for the three rate constants involved. It provides for the U-shape of the K(m) vs. T profile and allows for the derivation of an equation for identifying its inversion point. Modeling of V(max) and V(min) (reaction velocity under conditions of substrate saturation and of dilution, K(m)>>[S], respectively) is also included. An expression was formulated for predicting the "critical temperature," T(C), corresponding to the low-temperature break in Arrhenius lines. Using existing K(m) data from literature, concerning a variety of E-S systems, our modeling proved to be highly satisfactory. Our own experiments show that glucose uptake by rat brain synaptosomes can be regarded as a special case of basically the same kinetic scheme, and that the U-shaped temperature modulation of apparent K(m) for glucose conversion is also in full agreement with our kinetic modeling. These experiments indicate that positive thermal modulation, although based on intrinsic kinetic properties of the underlying E-S system, may have an adaptive role in endotherms as well, linked, however, to their tolerance to hypothermia.

Ca(2+)-mobilizing endothelin-A receptors inhibit voltage-gated Ca(2+) influx through G(i/o) signaling pathway in pituitary lactotrophs.

In excitable cells, receptor-induced Ca(2+) release from intracellular stores is usually accompanied by sustained depolarization of cells and facilitated voltage-gated Ca(2+) influx (VGCI). In quiescent pituitary lactotrophs, however, endothelin-1 (ET-1) induced rapid Ca(2+) release without triggering Ca(2+) influx. Furthermore, in spontaneously firing and depolarized lactotrophs, the Ca(2+)-mobilizing action of ET-1 was followed by inhibition of spontaneous VGCI caused by prolonged cell hyperpolarization and abolition of action potential-driven Ca(2+) influx. Agonist-induced depolarization of cells and enhancement of VGCI upon Ca(2+) mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin (PTX). Activation of adenylyl cyclase by forskolin and addition of cell-permeable 8-bromo-cAMP did not affect ET-1-induced sustained inhibition of VGCI, suggesting that the cAMP-protein kinase A signaling pathway does not mediate the inhibitory action of ET-1 on VGCI. Consistent with the role of PTX-sensitive K(+) channels in ET-1-induced hyperpolarization of control cells, but not PTX-treated cells, ET-1 decreased the cell input resistance and activated a 5 mM Cs(+)-sensitive K(+) current. In the presence of Cs(+), ET-1 stimulated VGCI in a manner comparable with that observed in PTX-treated cells, whereas E-4031, a specific blocker of ether-a-go-go-related gene-like K(+) channels, was ineffective. Similar effects of PTX and Cs(+) were also observed in GH(3) immortalized cells transiently expressing ET(A) receptors. These results indicate that signaling of ET(A) receptors through the G(i/o) pathway in lactotrophs and the subsequent activation of inward rectifier K(+) channels provide an effective and adenylyl cyclase-independent mechanism for a prolonged uncoupling of Ca(2+) mobilization and influx pathways.