Esther Zoref-Shani

Protein kinase C‐ε is involved in the adenosine‐activated signal transduction pathway conferring protection against ischemia‐reperfusion injury in primary rat neuronal cultures

Adenosine activates a signal transduction pathway (STP) in the heart and the brain, conferring protection against ischemia‐reperfusion insult. Activation of protein kinase C (PKC), probably mainly PKC‐ε, has been demonstrated to be part of the heart STP, but its role in the neuronal pathway is less clear. Here, we provide proof for the participation of PKC‐ε in the neuronal adenosine‐activated STP. Primary rat neuronal cultures were exposed to chemical ischemia by iodoacetate, followed by reperfusion. The cultured neurons were protected against this insult by activation of the adenosine mechanism, by N6‐(R)‐phenylisopropyladenosine [R(–)‐PIA], a specific A1 adenosine receptor agonist. Exposure of the cultures to bisindolylmaleimide I, a highly selective PKC inhibitor, abrogated the protection. The exposure of the cultures to R(–)‐PIA was found to result in phosphorylation (activation) of PKC‐ε. Furthermore, insertion into the cells of a specific peptide inhibitor of PKC‐ε translocation (εV1‐2), also abrogated the protection conferred by R(–)‐PIA. These results demonstrate that activation of PKC‐ε is a vital step in the neuronal adenosine‐activated STP.

Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures

The effect of opening and of blocking of ATP-sensitive potassium (K(ATP)) channels on the short-term capacity of neurons to resist ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to metabolic poisoning by iodoacetic acid (150 microM, 150 min), followed by reperfusion (1 h). The metabolic poisoning resulted in a marked decrease in cellular ATP content (from 65.3 +/- 13.4 to 21.6 +/- 11.7 nmole/mg protein), simulating an ischemia, or hypoxia-induced condition of energy crisis. The degree of neuronal damage was assessed by the trypan blue exclusion test. Exposure of the neurons to the channel-opener cromakalim (10 microM; 15 min), prior to the insult, induced resistance, which could be abolished by the specific channel blocker glibenclamide (2 microM). Glibenclamide also abolished the protection acquired by preconditioning of the neurons with iodoacetate (IA; 100 microM), the adenosine A1 agonist N6-(R)-phenylisopropyladenosine (R-PIA; 100 microM), or with the protein kinase C (PKC) activator 1,2 dioctanoyl-rac-glycerol (DOG; 1 microM). The results indicate that in the neurons, opening of the K(ATP) channels confers protection against an ATP-depleting crisis, and suggest that the protective effects induced by adenosine and by activation of PKC, are mediated by the opening of these channels.

Antiproliferative and differentiating effects of benzodiazepine receptor ligands on B16 melanoma cells

In this study, we evaluated the effect of several ligands active at the central-type and peripheral-type benzodiazepine receptor (BzR) (clonazepam, diazepam, PK11195 and Ro5-4864) on the growth and differentiation of B16 melanoma cells. All tested BzR ligands were able to suppress proliferation of the cells at the micromolar range and in a concentration-dependent manner. However, agents selectively active at the peripheral-type BzR (PK11195 and Ro5-4864) exhibited more potent antiproliferative activity. In addition, the BzR ligands were demonstrated to affect the cell cycle by reducing the percent of cells in the S phase and increasing the percent in the G2/M phase. BzR ligands induced cellular phenotypic alterations, which have been previously shown to be associated with melanoma cell differentiation. These alterations included: marked morphological changes, enhancement of melanogenesis, lipid accumulation and increase in the activity of gamma glutamyl transpeptidase. All BzR ligands induced a marked reduction in the concentration of UTP and most of them did the same in GTP and CTP, while ATP levels were not significantly altered. In summary, BzR ligands (clonazepam, diazepam, PK11195 and Ro5-4864) were found to exert antitumor effects in B16 melanoma cells. These findings encourage further studies of a possible therapeutic potential of BzR ligands in treatment of melanoma.

Oxidative stress activates transcription factor NF-κB-mediated protective signaling in primary rat neuronal cultures

Activation of transcription factor nuclear factor-κB (NF-κB) can result in enhanced de novo synthesis of both proteins that confer protection and those that cause death. The present study was undertaken to clarify in primary neuronal cultures the consequences of the oxidative stress-induced activation of NF-κB and mediation of death or survival signals. The neuronal cultures were exposed to chemical ischemia (iodoacetic acid), followed by reperfusion (I/R insult). This insult injured the neurons, as manifested in a 7- to 10-fold increase in LDH release, and decreased the cellular content of IκBα by 55–65 %, indicating NF-κB activation. The antioxidants LY231617, melatonin, and sodium salicylate and the antioxidant and inhibitor of NF-κB activation pyrrolidine dithiocarbamate, protected the neurons against the insult and prevented the decrease in cellular IκBα content. In contrast, inhibition of NF-κB translocation by SN50 in both uninsulated and insulted neuronal cultures resulted in a 2.9- and 2.4-fold increase in LDH release, respectively. The results indicate that the insult-induced oxidative stress activates transcription factor NF-κB associated with induction of protection and suggest that constitutive activation of NF-κB under physiological conditions acts to protect the neurons against physiological injury.

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice.

Abstract : Lesch‐Nyhan syndrome is a pediatric metabolic‐neurological syndrome caused by the X‐linked deficiency of the purine salvage enzyme hypoxanthine‐guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT‐deficient transgenic mice. The HGPRT‐deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT‐deficient cells. The following abnormalities were found : absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides ; 27.8% increase in the availability of 5‐phosphoribosyl‐1‐pyrophosphate ; 9.4‐fold acceleration of the rate of de novo nucleotide synthesis ; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine) ; enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2‐fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT‐deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch‐Nyhan syndrome.

Opening of KATP channels is mandatory for acquisition of ischemic tolerance by adenosine

Binding of adenosine to neuronal adenosine receptors activates a signal transduction pathway (the adenosine mechanism), leading to a temporary ischemic tolerance. We have demonstrated before that induction of this mechanism in primary rat neuronal cultures, by activation of adenosine receptors, or by activation of protein kinase C (PKC), confers a wide time window of ischemic tolerance, lasting up to 72 h, the early (immediate) part of which depends on opening of KATP channels (glibenclamide sensitive). Here we demonstrate that the entire duration of the ischemic tolerance conferred by activation of the adenosine mechanism depends on opening of the KATP channels. Thus, opening of the KATP channels appears to be a mandatory step in the adenosine mechanism, leading to the creation of the wide time window of ischemic tolerance.

Characterization of the Alterations in Purine Nucleotide Metabolism in Hypoxanthine‐Guardne Phosphoribosyltransferase‐Deficient Rat Neuroma Cell Line

Abstract: A rat neuroma cell line (B103 4C), deficient of hypoxanthine‐guanine phosphoribosyltransferase (HGPRT), was utilized as a model tissue in search for the biochemical basis of the Lesch‐Nyhan syndrome (LNS). The HGPRT‐deficient neurons exhibited the following properties: an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92% and 0.69% of normal, respectively); a significant increase in the availability of 5′‐phosphoribosyl‐1‐pyrophosphate; a three‐ to fourfold acceleration of the rate of de novo nucleotide synthesis; a normal excretion of xanthine, but 15‐fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purine nucleotide content, including the absence of 5‐amino‐4‐imidazole carboxamide nucleotides (Z‐nucleotides), but enhanced turnover of adenine nucleotides (loss of 86% of the radioactivity of the prelabeled pool in 24 h, in comparison to 73% in the normal line), and an elevated UTP content. The results suggest that, under physiological conditions, guanine salvage does not occur in the normal neurons, but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool. The finding of the normal profile of purine nucleotides in the HGPRT‐deficient neurons indicates that the lack of hypoxanthine salvage is adequately compensated by the enhanced de novo nucleotide synthesis. These results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z‐nucleotide accumulation, occurs in HGPRT‐deficient neurons and that these are etiological factors causing the neurological abnormalities in LNS. On the other hand, the results point to the possibility that elevated hypoxanthine concentration in the brain may have an etiological role in the pathogenesis of LNS.

Reactive oxygen species play an important role in iodoacetate-induced neurotoxicity in primary rat neuronal cultures and in differentiated PC12 cells

The role of reactive oxygen species in the pathogenesis of the neurotoxicity associated with ischemia-reperfusion, was investigated in a model of primary rat neuronal cultures and of differentiated PC12 cells, subjected to chemical ischemia by iodoacetic acid (IAA, 2.5 h) followed by a short period of reperfusion (1 h). The injury to the cells was assessed by lactate dehydrogenase (LDH) release into the culture media. The PC12 cells exhibited relative resistance to IAA cytotoxicity. Therefore these cells were studied at a 4-fold higher IAA concentration (400 microM instead of 100 microM for the neurons). The injury to both cell types was significantly greater in the short post-insult reperfusion (PIR) period than during the insult period. The presence, during the combined insult and PIR periods, of alpha-tocopherol (100 microM), melatonin (1 mM) and LY231617 (5 microM), conferred to both cell types considerable protection against the injury occurring during the insult and during the PIR periods (assessed separately). Superoxide dismutase (SOD; 500 IU/ml) conferred protection to the neurons, but not to the PC12 cells. When exposure to the antioxidants was limited to the short (15 min) pre insult period, only LY231617 conferred protection. In the neurons the protection occurred only during the insult period, whereas in the PC12 cells during both the insult and PIR periods. When the exposure to the antioxidants was limited to the PIR period, only SOD conferred protection and only in the neuronal cultures. These findings suggest that neuronal damage caused during ischemia-reperfusion can be diminished markedly by co-presence of antioxidants during the insult period. Certain antioxidants may protect the neurons even when present only before or after the insult.

DEVELOPMENTAL CHANGES IN PURINE NUCLEOTIDE METABOLISM IN CULTURED RAT ASTROGLIA

The present study was conducted in order to clarify the role of the glia in brain purine metabolism. This, in connection with the clarification of the etiology of the neurological manifestations associated with some of the inborn errors of purine metabolism in man. Purine nucleotide content, the capacity for de novo and salvage purine synthesis and the activity of several enzymes of purine nucleotide degradation, were assayed in primary cultures of rat astroglia in relation to culture age. The capacity of the intact cells to produce purine nucleotides de novo exhibited a marked decrease with the culture age, but the activity of hypoxanthine‐guanine phosphoribosyltransferase (HGPRT), catalyzing salvage nucleotide synthesis, increased. Aging was also associated with a marked increase in the activity of the degradation enzymes AMP deaminase, purine nucleoside phosphorylase (PNP) and guanine deaminase (guanase). The activity of adenosine deaminase and of AMP‐5′‐nucleotidase, increased markedly during the first 17 days in culture, but decreased thereafter.

Decelerated rate of dendrite outgrowth from dopaminergic neurons in primary cultures from brains of hypoxanthine phosphoribosyltransferase-deficient knockout mice.

Lesch-Nyhan syndrome (LNS), caused by the complete deficiency of hypoxanthine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is still unclear. Evidence has accumulated indicating that it reflects dopamine deficiency associated with defective arborization of dopaminergic dendrites. We monitored the differentiation in vitro of dopaminergic neurons, cultured from HPRT-deficient knockout mice. The HPRT-deficient dopaminergic neurons exhibited a decelerated rate of outgrowth of dendrites in comparison to that of control neurons resulting, after 8 days in culture, in 32% smaller average total length of dendrites per neuron (P<0.025). The results suggest that the abnormal dendrite outgrowth in LNS reflects a defective developmental process.