Joel Horwitz

Peroxynitrite‐Mediated Inhibition of DOPA Synthesis in PC12 Cells

Abstract: Experimental evidence has implicated oxidative stress in the development of Parkinsons disease, amyotrophic lateral sclerosis, and other degenerative neuronal disorders. Recently, peroxynitrite, which is formed by the nearly diffusion‐limited reaction of nitric oxide with superoxide, has been suggested to be a mediator of oxidant‐induced cellular injury. The potential role of peroxynitrite in the pathology associated with Parkinsons disease was evaluated by examining its effect on DOPA synthesis in PC12 pheochromocytoma cells. Peroxynitrite was generated from the compound 3‐morpholinosydnonimine (SIN‐1), which releases superoxide and nitric oxide simultaneously. Exposure of PC12 cells to peroxynitrite for 60 min greatly diminished their ability to synthesize DOPA without apparent cell death. The inhibition was due neither to the formation of free nitrotyrosine nor the oxidation of DOPA by peroxynitrite. The inhibition in DOPA synthesis by SIN‐1 was abolished when superoxide was scavenged by the addition of superoxide dismutase. These data indicated that neither nitric oxide nor hydrogen peroxide generated by the dismutation of superoxide is responsible for the SIN‐1‐mediated inhibition of DOPA production. The inhibition of DOPA synthesis at high concentration of SIN‐1 persisted even after removal of SIN‐1. The inactivation of the tyrosine hydroxylase may be responsible for the significant decline in DOPA formation by peroxynitrite. Inactivation of tyrosine hydroxylase may be part of the initial insult in oxidative damage that eventually leads to cell death.

Mechanisms of cell death and neuroprotection by poloxamer 188 after mechanical trauma

The mechanisms of cell death and the progressive degeneration of neural tissue following traumatic brain injury (TBI) have come under intense investigation. However, the complex interactions among the evolving pathologies in multiple cell types obscure the causal relationships between the initial effects of the mechanical trauma at the cellular level and the long‐term dysfunction and neuronal death. We used an in vitro model of neuronal injury to study the mechanisms of cell death in response to a well‐defined mechanical insult and found that the majority of dead cells were apoptotic. We have previously reported that promotion of membrane repair acutely with the non‐ionic surfactant poloxamer 188 (P188) restored cell viability to control values at 24 h postinjury. Here, we showed that P188 significantly inhibits apoptosis and prevents necrosis. We also examined the role of mitogen‐activated protein kinases (MAPKs) in cell death. There was a rapid, transient activation of extracellular signal‐regulated kinases, c‐Jun N‐terminal kinase, and p38s after mechanical insult. Of these, activation of the proapoptotic p38 was the greatest. Treatment with P188 inhibited p38 activation; however, direct inhibition of p38 by SB203580, which selectively inhibits the activity of the p38 MAPK, provided only partial inhibition of apoptosis and had no effect on necrosis. These data suggest that multiple signaling pathways may be involved in the long‐term response of neurons to mechanical injury. Furthermore, that the membrane resealing action of P188 provides such significant protection from both necrosis and apoptosis suggests that acute membrane damage due to trauma is a critical precipitating event that is upstream of the many signaling cascades contributing to the subsequent pathology.

Guanine nucleotide regulatory proteins, Gq and Gi1/2, mediate platelet-activating factor-stimulated phosphoinositide metabolism in immortalized hippocampal cells.

Abstract: Platelet‐activating factor (PAF) may be a neuromodulator involved in neural cell differentiation, cerebral inflammation, and ischemia. The PAF receptor is a member of the G protein‐coupled receptor superfamily. In the present study, we sought to define the specific G protein(s) that mediate PAF‐stimulated phosphoinositide (PI) metabolism in an immortalized hippocampal cell line, HN33.11. PAF increased the production of 3H‐labeled inositol phosphates (IPs) with EC50 values of 1.2–1.5 nM. The effect of PAF on 3H‐IPs formation was completely blocked by the PAF antagonist BN 50739 at a concentration of 300 nM. Pertussis toxin pretreatment attenuated PAF‐stimulated 3H‐IPs production by 20–30% (p < 0.05). Consistent with a role for Gi1/2 in this response, antiserum against Gαi1/2 blocked the response to a similar degree. Pretreatment of permeabilized cells with Gαq/11 antiserum attenuated the response by 70% (p < 0.05), suggesting a role for Gq/11 in mediating the PAF response in this cell line. Stimulation with PAF increased [α‐32P]‐GTP binding to both Gαq and Gαi1/2 proteins. Moreover, specific [3H]PAF binding sites coprecipitated with Gαq and Gαi1/2 proteins. The results suggest that PAF‐stimulated PI metabolism in HN33.11 cells is mediated by both Gq and Gi1/2 proteins.

Effect of aging on A1-adenosine receptor-mediated inhibition of norepinephrine release in the rat heart.

Adenosine inhibits norepinephrine (NE) release from cardiac adrenergic nerves and reduces the postsynaptic beta-adrenergic mediated actions of NE, leading to decreased myocardial force of contraction. The actions of adenosine are mediated by pre- and postsynaptic adenosine A1 receptors (A1-AdoR). We reported that adenosine inhibition of postsynaptic beta-adrenergic receptor-mediated cyclic adenosine monophosphate (cAMP) production declines with age in male F344 rat hearts. In this study, cardiac synaptosomes, isolated intact adrenergic nerve terminals, were used to examine the effect of age on adenosine inhibition of NE release. Cardiac synaptosomes were prepared from the hearts of 6- and 24-month-old male F344 rats, loaded with [3H]NE, and placed in a superfusion system. [3H]NE release was induced by high [K+] exposure in the presence of varying concentrations of adenosine or the specific A1-AdoR agonist, N6-p-sulfophenyladenosine (SPA). [3H]NE release was significantly reduced in old rats compared with young rats. Inhibition of [3H]NE release by adenosine and SPA was significantly greater in young rats compared with old rats. The A1-AdoR antagonist, 8-(p-sulfophenyl)-theophylline, blocked the actions of adenosine on [3H]NE release, and the specific adenosine A2-receptor agonist, cyclopropylcarboxamidoadenosine, had no effect on [3H]NE release. Our data suggest that presynaptic A1-AdoR-mediated inhibition of NE release in the rat heart declines with age.

Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2

These experiments were designed to learn the role of bradykinin induced changes in intracellular Ca2+ in the activation of phospholipase D activity in PC12 cells. Ionomycin at a concentration of 0.1μM caused an increase in intracellular Ca2+ comparable to bradykinin, but had no effect on phospholipase D activity. Carbachol, ATP, and thapsigargin also increased intracellular Ca2+ but had no effect on phospholipase D activity. Increases in intracellular Ca2+ may be a necessary but not a sufficient factor in the activation of phospholipase D. To investigate this issue, the bradykinin induced increase in intracellular Ca2+ was blocked by preincubating the cells in Ca2+-free media plus EGTA or in media containing the intracellular Ca2+ chelator BAPTA/AM. These preincubations completely blocked the bradykinin induced increase in intracellular Ca2+ but only attenuated the bradykinin mediated activation of phospholipase D. Physiological increases in intracellular Ca2+ apparently do not mediate the effect of bradykinin on phospholipase D.

Measurements of Phospholipases A2, C, and D (PLA2, PLC, and PLD)

In order to be properly divisible, the cell membrane has to be remodeled and intracellular membranes must be converted into a vesiculated state prior to mitosis. Phospholipases A2, C, and D (PLA2, PLC, and PLD) are involved in regulatory events of intracellular mitogen signaling pathways. We describe here three methods for comprehensively assaying those phospholipases: 1) in vitro microassays, in which a radiolabeled substrate is exogenously added to cell lysates to measure the enzyme activity(ies); 2) immunocomplex assays, in which immunoprecipitation with a specific antibody is performed in order to study the contribution of a particular isoform within a family of enzymes; and 3) intact-cell or in vivo assays, in which cells are labeled with a radioactive substrate until steady state is reached. The uniqueness of the in vitro microassay method described here for the first time is that it allows the measurement of, in parallel, the activities of three phospholipases utilizing aliquots derived from the same biological sample. The approach for immunoprecipitation described in this chapter can be extrapolated to the study of a large array of enzyme isoforms. Finally, the intact-cell assays allow for the accurate measurement of receptor-mediated activation in vivo.

Membrane Integrity as a Therapeutic Target in Neural Cell Injury

The importance of cell membrane integrity for normal cell function and indeed survival is well established, yet the role of membrane disruption in cellular pathology is seldom considered except as a prelude to, or indication of, cell death. However, evidence from diverse fields strongly implicates membrane disruption as a key precipitating event in the pathological responses to various stimuli. Dynamic mechanical loading of neural cells produces an acute disruption of the plasma membrane as indicated by a rapid and transient release of LDH from the cytoplasm of injured cells. In this report, we show that this cellular level injury is not immediately fatal, but rather gives rise to a cascade of signaling events that lead to cell death in the long term. In our model, over 50% of the cells were dead at 24 hours post injury, the majority of which were apoptotic as assessed by the TUNEL assay using flow cytometry. Though many of the signaling pathways involved in this response to injury have been studied, the link between the initial membrane damage and the subsequent signaling is poorly understood. We report for the first time that treating injured neurons with an agent that promotes resealing of membrane pores can rescue the cells from both necrotic cell death and apoptosis at 24 hours post injury. Treatment with the nonionic surfactant, poloxamer 188 (P188), at 15 minutes post injury restored cell viability at 24 hours to control values. The role of the pro-apoptosis MAP kinase, p38, in cell death following injury was investigated using Western blot analysis. Activation of p38 was increased over 2-fold at 15 minutes post injury. P188 treatment at 10 minutes inhibited p38 activation. However, treatment with a specific inhibitor of p38 activation produced only a partial reduction in apoptosis and had no effect on necrotic cell death. These data suggest multiple signaling pathways are involved in the long term response of neurons to mechanical injury. Furthermore, the putative mechanism of action of P188 to promote membrane resealing suggests that the acute membrane damage due to trauma is a critical precipitating event lying upstream of the many signaling cascades that contribute to the subsequent pathology.Copyright

Measurements of Phospholipases A 2 , C, and D (PLA 2 , PLC, and PLD)

In order to be properly divisible, the cell membrane has to be remodeled and intracellular membranes must be converted into a vesiculated state prior to mitosis. Phospholipases A2, C, and D (PLA2, PLC, and PLD) are involved in regulatory events of intracellular mitogen signaling pathways. We describe here three methods for comprehensively assaying those phospholipases: 1) in vitro microassays, in which a radiolabeled substrate is exogenously added to cell lysates to measure the enzyme activity(ies); 2) immunocomplex assays, in which immunoprecipitation with a specific antibody is performed in order to study the contribution of a particular isoform within a family of enzymes; and 3) intact-cell or in vivo assays, in which cells are labeled with a radioactive substrate until steady state is reached. The uniqueness of the in vitro microassay method described here for the first time is that it allows the measurement of, in parallel, the activities of three phospholipases utilizing aliquots derived from the same biological sample. The approach for immunoprecipitation described in this chapter can be extrapolated to the study of a large array of enzyme isoforms. Finally, the intact-cell assays allow for the accurate measurement of receptor-mediated activation in vivo.

Characterization of neuronal cell injury and neuroprotective effect of Poloxamer

To better understand the cellular mechanisms of neuronal injury, it is very important to mimic mechanical loading conditions experienced by the cells in vitro. Mechanical force applied to cells outside may activate some downstream events inside the cells, which may further cause necrosis or apoptosis. In this study, we used a cell culture model, which is an idealized system to investigate cell injury at the cellular and molecular levels. We developed an injury model, which allows a precisely controlled mechanical stimuli and an easy quantification of cellular responses. A dynamically controlled shear stress was applied on PC2 cells and cell viability was assessed at 24 hours. Dynamic mechanical loading of cells produced graded levels of injury as assessed by long-term viability. We also used Poloxamer to demonstrate its neuroprotective effect on the injured cells. This system can be used to investigate further the mechanism of the injury and to assess various treatments of neuronal injury and subsequent degeneration.

Effect of mechanical trauma on neuronal cell viability

To understand the cellular mechanisms of neuronal injury, it is very important to mimic the mechanical loading conditions experienced by the cells in vivo. A cell culture model is an idealized system to investigate cell injury at the cellular and molecular levels. The authors developed a model that allows mechanical stimuli to be precisely controlled and cellular responses to be quantified. In this study, shear stress was dynamically applied to PC2 cells and cell viability assessed at 24 hours. Dynamic mechanical loading of cells produced graded levels of injury as assessed by long-term viability. This system can now be used to investigate further the mechanism of the injury and to assess various treatments of neuronal injury and subsequent degeneration.