Prostate Apoptosis Response-4 Mediates Trophic Factor Withdrawal-Induced Apoptosis of Hippocampal Neurons

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.     

Divergent Effects of Acute Depolarization on Somatostatin Release and Protein Synthesis in Cultured Fetal and Neonatal Rat Brain Cells

The influence of membrane depolarization on somatostatin secretion and protein synthesis by fetal and neonatal cerebrocortical neurons was studied. Cortical cells obtained by mechanical dispersion were maintained as monolayer cultures for 8 days. The ability of fetal cerebrocortical and hypothalamic cells to release immunoreactive somatostatin (IR-SRIF) was confirmed. Total protein synthesis was determined by the incorporation of [3H]phenylalanine into trichloroacetic acid-precipitable proteins. To study the effect of acute depolarization on protein synthesis, cells were incubated for 30 min with [3H]phenylalanine or [3H]leucine and the depolarizing agent. In fetal cerebrocortical cells, potassium (30 and 56 mM) decreased protein synthesis and RNA levels and increased IR-SRIF release. Depolarization by veratridine, a sodium channel activator, induced a similar effect. The effect of veratridine on IR-SRIF and protein synthesis was reversed by tetrodotoxin, a sodium channel blocker, or verapamil, a calcium channel blocker. These findings suggest that protein synthesis by cerebrocortical cells is decreased in fetal brain cells by membrane depolarization and is dependent on Na+ and Ca2+ entry into cells. In postnatal (day 7) cerebrocortical cells, depolarization induced by high potassium concentrations led to a concomitant increase in protein synthesis, RNA content, and somatostatin release. These findings indicate that depolarization of the cellular membrane is coupled to an increase in protein synthesis in neonatal, but not in fetal, dispersed brain cells.     

Excitation-contraction coupling in resistance mesenteric arteries: Evidence for NKCC1-mediated pathway

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na⁺, K⁺, 2Cl⁻ cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 μM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K⁺]_o-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1^-/- mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.     

Unexpected roles for cryptochrome 2 and phototropin revealed by high-resolution analysis of blue light-mediated hypocotyl growth inhibition

Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de-etiolation, the transformation of a dark-grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high-resolution studies of hypocotyl growth accomplished by computer-assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the first beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin-dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin-dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non-redundantly to anion-channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL-activation of phototropin influences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL-induced stomatal opening, do not affect any aspect of the growth inhibition within the first 120 min examined here, and NPQ1 does not affect the activation of anion channels.     

Refinement of the spectra of exon usage by combined effects of extracellular stimulus and intracellular factors

Finely tuned differential expression of alternative splice variants contributes to important physiological processes such as the fine-tuning of electrical firing or hearing frequencies; yet the underlying molecular basis for the expression control is not clear. The inclusion levels of four depolarization-regulated alternative exons were measured by RT-PCR in GH₃ pituitary cells under different conditions of stimulation and/or RNA interference of splicing factors. The usage of the exons was reduced by membrane depolarization to various extents and was differentially modulated by the knock-down of splicing factors hnRNP L, L-like, I (PTBP1) or K or their combinations. A spectrum of each exon's level was produced under six knock-down conditions and was significantly shifted by depolarization. When all these conditions were considered together, a more refined or expanded spectrum of exon usage was obtained for each of the four exons. As a proof of principle for the molecular basis of the fine-tuning of exon usage, we show in the cases of hnRNP L and LL that their differential effects through the same element or different combinations of RNA sequences by the same factor hnRNP L are critical. The results thus demonstrate that the combined effect of varying extracellular stimuli and intracellular factors/RNA sequences refines or expands the spectra of endogenous exon usage, likely contributing to the fine-tuning of cellular properties.     

Neuronal Activation Regulates the Expression of Opioid Receptors: Possible Role of Glial-Derived Factors and Voltage-Dependent Ion Channels

The previous observation that a continuous chemical depolarization of aggregating rat brain cells with KCl alters the expression of opioid receptors was examined in more detail. In contrast to its significant and converse effect on forebrain and hindbrain cells cultured in serum-containing medium, KCl had only a small and transient effect in serum-free cultures of both types. The basal receptor density in serum-free cultures was similar to the receptor density in KCl-treated serum-containing cultures, but medium conditioned by glial cells restored partially the effect of KCl in serum-free cultures. The effect of KCl in serum-containing forebrain cultures was enhanced by the voltage-dependent calcium channel blocker verapamil, and magnesium and cadmium had a similar, though smaller, effect. The sodium channel activator veratridine had a profound and dose-dependent inhibitory effect on the expression of the receptors in forebrain and hindbrain cultures, and tetrodotoxin blocked the veratridine effect. Information about the selectivity of the effect of neuronal activation on the various opioid receptor subtypes was obtained with the neuroblastoma X glioma hybrid M8 cells that possess only delta type opioid receptors. A Scatchard analysis of [3H]etorphine binding to these cells has shown that depolarization increased the Bmax, but had little, if any, effect on the affinity (KD) of the ligand to the receptors. The significance of depolarization and voltage-dependent sodium and calcium channels on the expression of different opioid receptor subtypes is discussed.