Changes In Second Messenger Systems Research Articles

Properties of cytotoxic peptide-formed ion channels

Cytotoxic peptides are relatively small cationic molecules such as those found 1) in venoms, e.g., melittin in bee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, and lycotoxin I and II in wolf spider; 2) in skin secretions (e.g., magainin I and II from Xenopus laevis, dermaseptin from frog, antimicrobials from carp) and cells of the immune system (e.g., insect, scorpion, and mammalian defensins and cryptdins); 3) as autocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic beta-cells, prion peptide fragment 106-126 [PrP-(106-126)], and amyloid beta-protein (AbetaP) cytotoxic to neurons; and 4) as designed synthetic peptides based on the sequences and properties of naturally occurring cytotoxic peptides. The small cytotoxic peptides are composed of beta-sheets, e.g., mammalian defensins, AbetaP, amylin, and PrP-(106-126), whereas the larger cytotoxic peptides have several domains composed of both alpha-helices and beta-sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modify cell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These two nonexclusive mechanisms of action lead to changes in second messenger systems that further augment the abnormal electrical activity and distortion of the signal transduction causing cell death.

Nogo-A, a potent inhibitor of neurite outgrowth and regeneration.

The lack of regrowth of injured neurons in the adult central nervous system (CNS) of higher vertebrates was accepted as a fact for many decades. In the last few years a very different view emerged; regeneration of lesioned fibre tracts in vivo could be induced experimentally, and molecules that are responsible for inhibition and repulsion of growing neurites have been defined. Mechanisms that link cellular phenomena like growth cone turning or growth cone collapse to intracellular changes in second messenger systems and cytoskeletal dynamics became unveiled. This article reviews recent developments in this field, focusing especially on one of the best characterised neurite out-growth inhibitory molecules found in CNS myelin that was recently cloned: Nogo-A. Nogo-A is a high molecular weight transmembrane protein and an antigen of the monoclonal antibody mAb IN-1 that was shown to promote long-distance regeneration and functional recovery in vivo when applied to spinal cord-injured adult rats. Nogo-A is expressed by oligodendrocytes in white matter of the CNS. With the molecular characterisation of this factor new possibilities open up to achieve structural and functional repair of the injured CNS.

Polychlorinated biphenyls and methylmercury act synergistically to reduce rat brain dopamine content in vitro.

Consumption of contaminated Great Lakes fish by pregnant women is associated with decreased birth weight and deficits in cognitive function in their infants and children. These fish contain many known and suspected anthropogenic neurotoxicants, making it difficult to determine which contaminant(s) are responsible for the observed deficits. We have undertaken a series of experiments to determine the relevant toxicants by comparing the neurotoxic effects of two of these contaminants--polychlorinated biphenyls (PCBs) and methylmercury (MeHg)--both of which are recognized neurotoxicants. Striatal punches obtained from adult rat brain were exposed to PCBs only, MeHg only, or the two in combination, and tissue and media concentrations of dopamine (DA) and its metabolites were determined by high performance liquid chromatography. Exposure to PCBs only reduced tissue DA and elevated media DA in a dose-dependent fashion. Exposure to MeHg only did not significantly affect either measure. However, when striatal punches were simultaneously exposed to PCBs and MeHg, there were significantly greater decreases in tissue DA concentrations and elevations in media DA than those caused by PCBs only, in the absence of changes in media lactate dehydrogenase concentrations. Elevations in both tissue and media 3, 4-dihydroxyphenylacetic acid concentrations were also observed. We suggest that the significant interactions between these two toxicants may be due to a common site of action (i.e., toxicant-induced increases in intracellular calcium and changes in second messenger systems) that influences DA function. The synergism between these contaminants suggests that future revisions of fish-consumption guidelines should consider contaminant interactions.

Polychlorinated Biphenyls and Methylmercury Act Synergistically to Reduce Rat Brain Dopamine Content in Vitro

Consumption of contaminated Great Lakes fish by pregnant women is associated with decreased birth weight and deficits in cognitive function in their infants and children. These fish contain many known and suspected anthropogenic neurotoxicants, making it difficult to determine which contaminant(s) are responsible for the observed deficits. We have undertaken a series of experiments to determine the relevant toxicants by comparing the neurotoxic effects of two of these contaminants--polychlorinated biphenyls (PCBs) and methylmercury (MeHg)--both of which are recognized neurotoxicants. Striatal punches obtained from adult rat brain were exposed to PCBs only, MeHg only, or the two in combination, and tissue and media concentrations of dopamine (DA) and its metabolites were determined by high performance liquid chromatography. Exposure to PCBs only reduced tissue DA and elevated media DA in a dose-dependent fashion. Exposure to MeHg only did not significantly affect either measure. However, when striatal punches were simultaneously exposed to PCBs and MeHg, there were significantly greater decreases in tissue DA concentrations and elevations in media DA than those caused by PCBs only, in the absence of changes in media lactate dehydrogenase concentrations. Elevations in both tissue and media 3, 4-dihydroxyphenylacetic acid concentrations were also observed. We suggest that the significant interactions between these two toxicants may be due to a common site of action (i.e., toxicant-induced increases in intracellular calcium and changes in second messenger systems) that influences DA function. The synergism between these contaminants suggests that future revisions of fish-consumption guidelines should consider contaminant interactions.

Alterations of second messenger systems in the rat brain after 6-hydroxydopamine lesions of the medial forebrain bundle

We studied the sequential changes in second messenger systems in the striatum and substantia nigra (SN) after 6-hydroxydopamine lesions of the medial forebrain bundle in rats. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks postlesion. [ 3H]Phorbol-12,13-dibutyrate (PDBu), [ 3H]forskolin and [ 3H]rolipram were used to label protein kinase C (PKC), adenylyl cyclase and calcium/calmodulin-independent cyclic-AMP phosphodiesterase, respectively. The degeneration of nigrostriatal pathway produced a significant increase in [ 3H]PDBu binding in the ventromedial part of the ipsilateral striatum from 2 to 8 weeks postlesion. In the contralateral side, [ 3H]PDBu binding showed a transient increase in the SN only 4 weeks after lesioning. [ 3H]Forskolin binding showed a significant increase in the ipsilateral and contralateral striatum from 2 to 4 weeks postlesion. In the ipsilateral SN, a significant increase in [ 3H]forskolin binding was observed at 4 weeks after lesioning. However, no significant change in [ 3H]forskolin binding was observed in the contralateral SN during postlesion. On the other hand, [ 3H]rolipram binding showed no conspicuous alteration in the brain during postlesion. These results demonstrate that rats made hemiparkinsonism by unilateral 6-hydroxydopamine injection have a significant increase in [ 3H]PDBu and [ 3H]forskolin binding in the striatum and/or SN, whereas no significant change in [ 3H]rolipram binding is observed in these areas during postlesion. Our findings also suggest that the increase in [ 3H]forskolin binding is more pronounced than that in [ 3H]PDBu binding in the brain after unilateral 6-hydroxydopamine injection. Thus, our studies may provide valuable information concerning degeneration of the nigrostriatal pathway such as Parkinson’s disease.

Repeated Exposure of Adult Rats to Aroclor 1254 Causes Brain Region-Specific Changes in Intracellular Ca2+Buffering and Protein Kinase C Activity in the Absence of Changes in Tyrosine Hydroxylase

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic.In vitrostudies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca2+homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBsin vivoby dosing adult male Long–Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca2+buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca2+buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4(total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1)in vivoexposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed afterin vitroexposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels.

Predicting In vivo Toxicity

The objective of toxicity testing is to generate information that will help establish the safety of new products. Successful new in vitro tests will identify those endpoints and cell systems which provide evidence of pathologic change that can be used in determining safety. Chronic in vivo toxicity studies usually document adverse changes that are indicative of cell death in a target organ. For example, leakage of tissue specific enzymes is indicative of membrane damage and correlated with microscopic observation of cell death. Tissues, like the liver, can mount an effective regenerative response to moderate cell loss, but cell proliferation may be accompanied by the possibility of carcinogenesis. The balance between cell death and cell proliferation can make the difference between recovery, organ failure or tumour induction. Consequently, in vitro studies of toxicity have often focused on cell death as an endpoint. Molecular events preceding the irreversible commitment of a cell to die or proliferate are currently being investigated as additional potentially useful endpoints for in vitro toxicity studies. As molecular mechanisms become better understood, the number of potential endpoints for in vitro toxicity testing has greatly increased. However, at the earliest stages of interaction with the cell, it is difficult to predict whether changes induced by the xenobiotic are reversible (pharmacologic) or irreversible (pathologic) change. Changes in second messenger systems, binding to receptors, or early gene induction may explain the induction of toxicity by agents previously studied in vivo, but are potentially reversible changes that may not be useful for predicting the safety of untested products. Later changes that can be more easily identified as irreversible may provide more useful endpoints. One example is the activation of caspases in the cell as a precursor to apoptosis.

Effect of nitric oxide synthase inhibitor on age-related changes in second messenger systems and calcium channels in rats

We investigated the effects of age and nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on protein kinase C (PKC), adenylyl cyclase, calcium/calmodulin-independent cyclic-AMP phosphodiesterase (cyclic-AMP PDE) and voltage-dependent L-type calcium channels in Fischer rat brain using autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]forskolin, [3H]rolipram and [3H]PN200-110 were used to label PKC, adenylyl cyclase, cyclic-AMP PDE and calcium channels, respectively. [3H]Forskolin binding significantly decreased in the striatum, hippocampal CA3 sector, dentate gyrus, hilus, thalamus, substantia nigra and cerebellum of 24-month-old (aged) rats, as compared with 6-month-old (adult) animals. [3H]Rolipram binding also showed an age-related reduction in the thalamus and cerebellum in rats. In contrast, no age-related changes were observed in [3H]PDBu and [3H]PN200-110 binding in the rat brain. Chronic treatment with L-NAME (5 mg/kg, once a day for 4 weeks) showed no significant changes in [3H]PDBu, [3H]rolipram and [3H]PN200-110 binding in aged rat brains. However, this treatment significantly increased age-related decreases in [3H]forskolin binding in the frontal cortex; striatum and hippocampal CA1 sector in rats. The results demonstrate that [3H]forskolin binding in the rat brain is more susceptible to aging processes than [3H]PDBu, [3H]rolipram and [3H]PN200-110 binding. Furthermore, our study shows that chronic treatment with NO inhibitor increases the age associated changes in [3H]forskolin binding in most brain areas of aged rats. These findings suggest that NO may play a key role in the regulation of adenylyl cyclase system during aging processes.

Modification of calcium flux of twitch skeletal muscle in mice subjected to 20% body surface area burn.

One systemic effect of burn trauma is skeletal muscle weakness. This is the result of changes in second messenger systems involving calcium (Ca2+). Kinetic analysis of Ca2+ from cellular compartments of skeletal muscle of mice that were subjected to small burn injury (20% body surface area) was performed. Muscles of the burn group showed an increased 45Ca2+ uptake maxima compared with those of time-matched control groups. Also, 45Ca2+ efflux analysis showed a lack of differentiation between electrically stimulated tissues and nonstimulated tissues that was easily observed in time-matched control groups. This lack of differentiation indicated changes in intracellular compartmentation. It can be speculated that burn trauma may have a regulatory role in the excitation-contraction coupling mechanism by altering myoplasmic levels of Ca2+ even before skeletal muscle dysfunction occurs. These findings suggest that agents capable of controlling myoplasmic Ca2+ levels may aid in alleviating chronic skeletal muscle dysfunction in patients with burns.

Growth factors and oncogenes in breast cancer

The growth of normal breast epithelial cells is regulated by a complex interacting system of polypeptide factors and by steroid hormones. The cells respond to these factors through receptors which generate mitogenic and other intracellular signals. These second messengers provoke complex responses which may ultimately result in DNA replication and cell division. A comparison of normal cells and tumour cells, either in culture or from primary tumour biopsies, has revealed differences in growth factor and growth factor receptor expression. Such changes may represent aspects of the process of malignant transformation. In addition some evidence suggests that changes in second messenger systems may also occur. Finally several changes in nuclear oncogenes have been observed in breast cancers. It has been proposed that changes in the nuclear oncogenes, perhaps involving the loss of function of tumour suppressor genes, may allow cells to enter the cell cycle. Changes in growth factors, their receptors or intracellular second messenger systems may stimulate unregulated growth. The combination of these events provide a model for the process of carcinogenesis.

New tools for the localization of second messenger systems.

1. Probes available for the localization of components of second messenger systems include G-protein oligonucleotides which have been used to produce cDNA probes to label G-protein mRNA by in situ hybridization histochemistry. 2. Enzymes involved in second messenger responses have been labelled with [3H]-forskolin (Gs-linked adenylate cyclase), [3H]-cAMP (cAMP-dependent protein kinase) and [3H]-PDBu2 (protein kinase C). 3. [3H]-Inositol 1,4,5 trisphosphate labels a site on sarcoplasmic reticulum believed to trigger Ca2+ release. 4. These probes allow the comparison of location of receptors and second messengers and the examination of changes in second messenger systems with drug treatment and disease.

Stimulation of guanylate cyclase activity and reduction of adenylate cyclase activity by granulocyte-macrophage colony-stimulating factor in human blood neutrophils.

Human neutrophils were incubated with granulocyte-macrophage (GM)-CSF and examined for changes in second messenger systems. Twofold increases in cGMP but not cAMP were measured after 5 to 20 min with 100 U/ml GM-CSF. Guanylate cyclase activities in membrane and cytosol fractions were increased to the same extent whether measured in the presence of Mg2+ or Mn2+, or in the cytosol with Mg2+ + N-methyl-N'-nitro-N-nitroso-guanidine. Kinetic studies of the cytosol enzyme showed no changes in the Km values for Mg2+ and Mn2+dependent guanylate cyclase activities (0.91 and 0.022 mM, respectively), whereas Vm values were increased after treating intact cells with GM-CSF. Two peaks of guanylate cyclase activity were observed, one at 10 and another at 60 min after adding 100 U/ml GM-CSF, whereas only one peak at 5 min occurred with 1 U/ml. Adenylate cyclase activity was reduced by nearly 50% after adding 100 U/ml GM-CSF for 10 to 30 min. These effects were also seen in the presence of several hormonal and nonhormonal adenylate cyclase stimulators. In contrast, small increases in adenylate cyclase activity occurred after adding 1 U/ml GM-CSF. In experiments to examine the pathway of guanylate cyclase activation by GM-CSF, we observed no changes in inositol phosphates, intracellular calcium ion, or cytosolic protein kinase C. The augmentation of chemotactic peptide-induced superoxide production by GM-CSF concentrations, may be related to the effects of the higher levels of GM-CSF to stimulate late increases in guanylate cyclase or decreases in adenylate cyclase.