Emma R. Jakoi

Acute brain death alters left ventricular myocardial gene expression

OBJECTIVES The depressed myocardial function observed in brain dead organ donors has been attributed to massive sympathetic discharge and catecholamine cardiotoxicity. Because elevated catecholamines are associated with altered myocardial gene expression, we investigated whether acute brain death from increased intracranial pressure alters the expression of myocardial gene products important in contractility. METHODS A balloon expansion model was used to increase intracranial pressure in rabbits (n = 22). At timed intervals after brain death, mean arterial pressure, heart rate, electrocardiograms, histologic myocardial injury, and systemic catecholamines were assessed. Messenger RNA levels encoding myofilaments, adrenergic receptors, sarcoplasmic reticulum proteins, transcription factors, and stress-induced programs were measured with blot hybridization of total left ventricular RNA. RESULTS Increased intracranial pressure induced an immediate pressor response that temporally coincided with diffuse electrocardiographic ST segment changes. Systemic epinephrine and norepinephrine levels concurrently increased (5- to 8-fold within 1 minute), then fell below baseline within 2 hours, and remained depressed at 4 hours. By 1 hour, histologic injury was evident. Four hours after the induction of increased intracranial pressure, levels of messenger RNA-encoding skeletal and cardiac alpha-actins, egr-1, and heat shock protein 70 were significantly increased. Sham-operated animals did not exhibit these changes. CONCLUSIONS Select changes in myocardial gene expression occur in response to increased intracranial pressure and implicate ventricular remodeling in the myocardial dysfunction associated with acute brain death.

Excitatory amino acid receptor activation produces a selective and long-lasting modulation of gene expression in hippocampal neurons

Activation of excitatory amino acid (EAA) receptors in cultured hippocampal neurons causes down-regulation of the protein ligatin, a receptor for phosphoglycoproteins and a marker protein for membrane-vesicle transport systems. This reduction occurs at both physiologic and excitotoxic levels of glutamate stimulation and is accompanied by a significant decrease in steady state levels of ligatin mRNA. Reduction in ligatin mRNA occurs within 60 min and persists 24 h later. Steady state levels of mRNAs encoding cyclophilin, an ubiquitous cytosolic protein, and neuron specific-enolase (N-SE) are not diminished by glutamate receptor activation, demonstrating that down-regulation of ligatin mRNA was not a result of general catabolism. Further, this reduction in ligatin mRNA occurred without induction of HSP 70. Pharmacological studies using selective antagonists and agonists indicate that this down-regulation of ligatin gene expression is predominantly mediated by the N-methyl-D-aspartate (NMDA) subclass of EAA receptors and that Ca2+ is required. This is the first report that EAA receptor activation in hippocampal neurons can pretranslationally down-regulate gene expression in a rapid and long-lasting manner under physiologic, as well as cytotoxic conditions. The data support the hypothesis that modulation of neuronal gene expression may represent a molecular mechanism mediating some of the long-lasting functional and pathophysiological effects of EAA on cell function.

GABAA α2 mRNA levels are decreased following induction of spontaneous epileptiform discharges in hippocampal-entorhinal cortical slices

Exposure of hippocampal slices to Mg2+ free media (0 Mg) has been shown to trigger full production of stimulus-induced seizure activity after restoration of physiological conditions [1]. In the present study employing hippocampal entorhinal cortical slices (HEC), spontaneous epileptiform discharges (SEDs) were induced using 0 Mg treatment following the return of the slices to physiological conditions. To evaluate the effect of sustained epileptiform activity on gene expression in this HEC slice preparation, changes in mRNA levels of the GABAA alpha 1 and alpha 2 and beta CaM Kinase II subunits were measured using in situ hybridization. HEC slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence or absence of Mg2+ for 3 h, then placed in oxygenated ACSF containing Mg2+ for up to 3 h. Control slices were maintained in Mg2+ containing ACSF for up to 6 h. Recurrent SEDs were observed in 0 Mg pre-treated slices while no epileptiform discharges were seen in control slices. Following induction of SEDs by 0 Mg pre-treatment, a significant decrease in mRNA encoding GABAA alpha 2 was found in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus for up to 3 h after treatment. Levels of mRNA for GABAA alpha 1 and beta CaM Kinase II were not affected. The results document a decrease in GABAA alpha 2 gene expression following the induction of SEDs in the HEC slice preparation and suggest that rapid changes in neuronal gene expression may contribute to long lasting excitability changes associated with the induction of epilepsy.

Glutamate receptor activation regulates mRNA at both transcriptional and posttranscriptional levels.

Abstract: Previous studies from this laboratory have demonstrated that extracellular calcium entry through the NMDA subtype of glutamate receptors in hippocampal neurons selectively down‐regulated ligatin gene expression in a rapid and long‐lasting manner. Here we investigated the molecular mechanism that underlies this phenomenon. We demonstrate that glutamate receptor activation transiently increased the transcriptional activity of the ligatin gene and simultaneously shortened the half‐life of its message. Using nuclear run‐on assays and northern analyses of total RNA from α‐amanitin‐treated cells, we measured the effects of glutamate on the transcriptional activity and mRNA stability of the ligatin gene. The transcriptional activity of ligatin was found to be transiently increased (1.4‐fold) 20 min after the addition of glutamate, with a return to basal levels by 60 min. Thus, the glutamate‐dependent decrease in ligatin message could not be explained by a decline in its synthesis. Instead, concurrent with transcriptional up‐regulation, glutamate shortened the half‐life of the ligatin message from 10 h to 58 min, leading to a net decrease (0.7‐fold) in its steady‐state levels by 60 min. This posttranscriptional destabilization of ligatin mRNA was mimicked by the translation inhibitor, cycloheximide, but not by puromycin. This finding indicated that the stability of ligatin mRNA was translation independent and distinguished this posttranscriptional regulatory mechanism from those previously described. Moreover, using in situ hybridization and confocal microscopy, we showed that control of message stability occurred both in the cell body and in the dendritic regions distant from the nucleus. The selective glutamate‐induced changes in the synthesis and stability of mRNA indicated that transcriptional and posttranscriptional regulation of gene expression may be part of the normal receptor‐mediated regulatory program of plasticity and may play an important role in local modulation of neuronal phenotype by neurotransmitters. Our results provide the first description of a receptor‐modulated, calcium‐dependent mechanism that rapidly destabilizes mRNA in neurons.

Phenylephrine induces delayed cardioprotection against necrosis without amelioration of stunning

BACKGROUND Alpha-adrenergic stimulation induces protection in reperfused ischemic (I/R) myocardium 24 hours later. We tested the hypothesis that phenylephrine improves dysfunction after global I/R by limiting cell death not stunning. METHODS Rabbits were pretreated with either phenylephrine or vehicle. Twenty-four hours later, isolated hearts underwent either 45 (infarction protocol) or 20 minutes (stunning protocol) of global ischemia before 2 hours of reperfusion (n = 6 per group). Cell death was determined by triphenyl tetrazolium chloride staining (infarction) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (apoptosis). RESULTS Compared with vehicle, phenylephrine pretreatment improved post-I/R-developed pressures in hearts after infarction (53.2 +/- 4.0 vs 35.8 +/- 4.1 mm Hg, p = 0.01) but not stunning protocol (64.3 +/- 8.9 vs 57.7 +/- 6.2 mm Hg, p = NS). The improved developed pressure was due to better diastolic recovery. Systolic pressures were similar between groups. Phenylephrine markedly decreased infarction (9.0 +/- 1.9% vs 40.8 +/- 1.8% for vehicle, p < 0.001) and TUNEL-positive staining. Stunned hearts of either group had less than 3% infarction and no apoptosis. CONCLUSIONS Phenylephrine pretreatment 24 hours before global I/R improves function by limiting infarction but not stunning.

Post-transcriptional regulation of gene expression in hippocampal neurons by glutamate receptor activation

Previous work from this laboratory has documented that glutamate receptor activation and extracellular calcium entry into hippocampal neurons caused a long-lasting down-regulation of ligatin mRNA and protein. Here, we investigated whether glutamate reduced ligatin mRNA levels by decreasing the transcriptional activity of the gene and/or by regulating post-transcriptional RNA processing steps including mRNA stability. Using nuclear run-on assays, it was demonstrated that transcriptional activity of the ligatin gene was not significantly decreased after glutamate receptor activation. Further, Northern analysis of RNA from neurons maintained in the presence of the transcription inhibitor, alpha-amanitin, showed that glutamate shortened the half life of the ligatin message from 10 h to 58 min. This post-transcriptional destabilization of ligatin mRNA was mimicked by NMDA, dependent on Ca2+, blocked by MK801, and not affected by AMPA and kainic acid, indicating that message stability was governed by changes in intracellular calcium. Moreover, using in situ hybridization and confocal microscopy, we showed that glutamate and NMDA decreased ligatin message within dendritic and somal regions without increasing nuclear levels. These findings demonstrated that glutamate receptor activation altered neuronal gene expression posttranscriptionally by destabilizing mRNA. Our data suggest that post-transcriptional regulation of gene expression may be part of the normal receptor mediated regulatory program of plasticity and provides the first description of a glutamate receptor-modulated, calcium-dependent mechanism which rapidly destabilizes mRNA in neurons.

Orotic acid improves left ventricular recovery four days after heterotopic transplantation

Orotic acid accelerates compensatory myocardial hypertrophy after regional ischemia and improves left ventricular function acutely after global ischemia. In this study, the effect of orotic acid on left ventricular function was investigated 4 days after global ischemia (75 minutes, 21 degrees C) using heterotopically transplanted rabbit hearts (n = 18). Experimental animals received daily 100-mg/kg doses of intraperitoneally administered orotic acid, starting 1 day before transplantation, and showed a threefold increase in the serum level of orotic acid by 4 days. After 1 hour of reperfusion, the developed pressure was equally depressed in both the control and experimental groups; however, 4 days later, the developed pressure in control animals was decreased by 3 +/- 3 mm Hg (versus the developed pressure measured at 1 hour) while the developed pressure in experimental animals was significantly increased by 25 +/- 8 mm Hg. Heterotopically transplanted hearts manifested diminished systolic function (stemming from ischemia and unloading) as well as decreased expression of adult myosin. Because orotic acid has been observed to produce an increase in protein synthesis in other models, we investigated whether this improvement in systolic function resulted from an orotic acid-mediated augmentation (or preservation) or normal adult myosin expression. Both orotic acid-treated and untreated hearts manifested decreased expression of the beta-myosin heavy chain protein and steady-state messenger RNA levels. Because function improved with decreased beta-myosin heavy chain expression, an alternate mechanism underlying orotic acid-mediated improvement in function is implicated. Nevertheless, orotic acid may be a therapeutic agent facilitating long-term recovery from global ischemia.

Distribution of anionic sites on surfaces of retinal cells: A study using cationized ferritin

The nonspecific ligand polycationized ferritin was used to visualize the topographical distribution of anionic sites present on surfaces of embryonic neural retinal cells. Binding of the ligand to isolated plasma membranes and to surfaces of intact and dissociated retinal cells was observed to be in a hexagonal array as seen by electron microscopy. This hexagonal lattice consisted of ferritin bound equidistantly with a center-to-center spacing of 14.5 nm. Cationized ferritin also bound in lattices to surfaces of retinal cell aggregates formed in culture but appeared to be restricted to focal areas of contact between cells and not to cell type. No effect on cationized ferritin pattern and therefore on charge distribution was observed in retina treated with neuraminidase or trypsin suggesting the anionic sites were not due to sialic acid or to trypsin-sensitive residues. However, treatment of isolated membranes with EGTA randomized the distribution of bound cationized ferritin. The appearance of the lattice was independent of conditions used for incubation with cationized ferritin and for electron microscopy fixation, suggesting these ordered arrays were not artifactual. Because of its wide distribution among the various cell types present in retina, a generalized function for this differentiated membrane in cell-cell contact is suggested.

Disruption of mRNA-RNP formation and sorting to dendritic synapses by antisense oligodeoxynucleotides

Publisher Summary Cellular differentiation requires a heterogeneous distribution of proteins to establish and maintain regions of specialized function. Two mechanisms have been identified that provide for spatial localization of proteins: the first involves directed targeting of the polypeptide and the second utilizes sorting of the encoding mRNA for localized protein synthesis. Although the localization of mRNA is well established as an important mechanism for generating polarity during embryogenesis, it has only recently emerged as an important means for establishing regional differentiation in somatic cells, such as neurons, fibroblasts, and skeletal myoblasts, where the local regulation of function may be critical. This process involves the formation of ribonucleoprotein particles (RNP), which govern sorting and anchoring of the mRNA. In general, two components are involved: a cis-acting mRNA sequence(s) that encodes spatial information and transacting proteins that confer stability, govern translation, and mediate tethering to cytoskeletal elements and/or movement. Identities of the cis-acting and transacting components that mediate mRNA localization in somatic cells remain largely unknown.

Intercellular Adhesion among Developing Retinal Cells: A Role for Ligatin as a Baseplate

A topographic selectivity characterizes the adhesion of neural retina cells to optic tecta. Cell bodies from dorsal half-retina have been shown to adhere preferentially to ventral surfaces of optic tecta, while those from ventral half-retina prefer dorsal surfaces of tecta (Barbera et al., 1973; Marchase et al., 1975). Subsequent demonstrations of intraretinal differences in specificity (Gottlieb et al., 1976) have shown a pronounced gradient in adhesive preferences along the dorso-ventral axis of the developing retina.