Martha Kaloyianni

Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring

The neutral red lysosomal retention assay (NRR) of the haemocytes, and the acetylcholinesterase activity (AChE) in the haemolymph, the digestive gland, the gills and the mantle/gonad complex have been evaluated on mussels Mytilus galloprovincialis collected from Thermaikos and Strymonikos gulfs (northern Greece) in June and October 2001. The validity of performing the above core biomarkers is supported, firstly by their ability to respond to different pollution levels and, secondly, by the significant linear correlation among them. The evaluation of the micronuclei frequency (MN) has been performed in gill tissue and haemocytes of the same mussels and, according to the results, it needs more research in order its use as stress indices to be validated. In addition, the first results on cAMP levels in the gills, the mantle/gonad complex and the digestive gland, whose concentrations correlated to both, NRR and AChE introduce this signal transduction molecule as a new, promising biomarker.

Cadmium effects on ROS production and DNA damage via adrenergic receptors stimulation: Role of Na+/H+ exchanger and PKC

The objective of the present study was to elucidate the events that are involved in reactive oxygen species (ROS) production and DNA damage after adrenergic receptors stimulation by cadmium, in relation to cAMP, protein kinase C (PKC) and Na+/H+ exchanger (NHE). Cadmium (50 μM) caused increased levels of ROS with a concomitant increase in DNA damage in digestive gland of Mytilus galloprovincialis. Either the use of EIPA, a NHE blocker, or calphostin C, the inhibitor of PKC, reduced cadmium effects. Cells treated with α1-, α2-, β- and β1- adrenergic antagonists together with cadmium reversed cadmium alone effects, while the respective adrenergic agonists, phenylephrine and isoprenaline, mimic cadmium effects. Moreover, cadmium caused an increase in the levels of cAMP in digestive gland cells that were reversed after NHE and PKC inhibition as well as in the presence of each type of adrenergic antagonist. The different sensitivity of α1-, α2-, β-, β1- adrenergic receptors on ROS, cAMP production and DNA damage possibly leads to the induction of two signaling pathways that may be interacting or to the presence of a compensatory pathway that acts in concert with the α- and β- adrenergic receptors. In these signaling pathways PKC and NHE play significant role.

Biomarkers in marine mussels, Mytilus galloprovincialis, exposed to environmentally relevant levels of the pesticides, chlorpyrifos and penoxsulam

The present study examines the influence of environmentally relevant concentrations of two pesticides, chlorpyrifos and penoxsulam on mussel physiological status. For this reason, lysosomal membrane stability (LMS), reactive oxygen species (ROS), DNA damage, protein carbonylation (PCC) and antioxidant capacity (TAC) in hemaolymph and hemocytes of the mussels was measured. Mussels were exposed to a range of concentrations of the pesticides chlorpyrifos and penoxsulam and the response of animals to the destabilization of lysosomal membrane in hemocytes (LMS) was studied. Subsequently, the half maximal effective concentration (EC50) for both pesticides was calculated. The animals were subsequently exposed for 0, 1, 3, 5, 7, 15 and 30 days to 10 times less concentration than EC50 of each pesticide (0.05 μg/l) and changes in LMS, ROS, DNA damage, protein carbonylation and antioxidant capacity of mussels was evaluated. Our results showed a significant change in the response of mussels for all parameters tested after 30 days exposure, in relation to the controls. The pesticides at the environmental concentrations used induced changes to the animal physiology through causing oxidative stress and lysosomal abnormalities and their usage in the agriculture demands great care. In addition, the results show that ROS, DNA damage, protein carbonylation and antioxidant capacity could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors.

Cadmium induces both pyruvate kinase and Na+/H+ exchanger activity through protein kinase C mediated signal transduction, in isolated digestive gland cells of Mytilus galloprovincialis (L.).

SUMMARY The present study investigates the transduction pathway mediated by cadmium in isolated digestive gland cells of mussel Mytilus galloprovincialis. The effects of cadmium treatment on a key glycolytic enzyme, pyruvate kinase (PK), and on Na+/H+ exchanger activity were examined. Cadmium (50 μmol l–1) caused a significant elevation of intracellular pH (pHi) and a rise (176%) of Na influx relative to control values. The amiloride analogue, EIPA (20 nmol l–1), a Na+/H+ exchanger blocker, together with cadmium, significantly reduced the effect of treatment by cadmium alone on both Na+ influx and pHi. In addition, PK activity was significantly increased after treatment with cadmium. PK activity was inhibited after treatment of cells with amiloride or EIPA together with cadmium. Moreover, phorbol-ester (PMA), a potent activator of protein kinase C (PKC), caused a significant rise in both pHi and PK activity, while staurosporine or calphostin C reversed both events. Adrenaline, isoprenaline and phenylephrine alone or together with cadmium also significantly increased the pHi and PK activity of isolated digestive gland cells. The latter effectors in combination with cadmium showed a synergistic effect on pHi and PK. These responses seem to be blocked by propranolol, metoprolol and prazosin. Our findings suggest a hormone-like effect of cadmium on digestive gland cells. The signal transduction pathway induced by cadmium involves the stimulation of PK, PKC and Na+/H+ exchanger in isolated digestive gland cells of Mytilus galloprovincialis.

The role of signalling molecules on actin glutathionylation and protein carbonylation induced by cadmium in haemocytes of mussel Mytilus galloprovincialis (Lmk).

SUMMARY This study investigated the role of Na+/H+ exchanger (NHE) and signalling molecules, such as cAMP, PKC, PI 3-kinase, and immune defence enzymes, NADPH oxidase and nitric oxide synthase, in the induction of protein glutathionylation and carbonylation in cadmium-treated haemocytes of mussel Mytilus galloprovincialis. Glutathionylation was detected by western blot analysis and showed actin as its main target. A significant increase of both actin glutathionylation and protein carbonylation, were observed in haemocytes exposed to micromolar concentration of cadmium chloride (5 μmol l–1). Cadmium seems to cause actin polymerization that may lead to its increased glutathionylation, probably to protect it from cadmium-induced oxidative stress. It is therefore possible that polymerization of actin plays a signalling role in the induction of both glutathionylation and carbonylation processes. NHE seems to play a regulatory role in the induction of oxidative damage and actin glutathionylation, since its inhibition by 2 μmol l–1 cariporide, significantly diminished cadmium effects in each case. Similarly, attenuation of cadmium effects were observed in cells pre-treated with either 11 μmol l–1 GF-109203X, a potent inhibitor of PKC, 50 nmol l–1 wortmannin, an inhibitor of PI 3-kinase, 0.01 mmol l–1 forskolin, an adenylyl cyclase activator, 10 μmol l–1 DPI, a NADPH oxidase inhibitor, or 10 μmol l–1 L-NAME, a nitric oxide synthase inhibitor, suggesting a possible role of PKC, PI 3-kinase and cAMP, as well as NADPH oxidase and nitric oxide synthase in the enhancement of cadmium effects on both actin glutathionylation and protein carbonylation.

Modulation of the Na+–H+ antiport activity by adrenaline on erythrocytes from normal and obese individuals

The effect of adrenaline on normal and obese human Na(+)-H(+) antiport (NHE 1) erythrocyte activity has been studied. Adrenaline increased both intracellular pH (pHi) and Na(+) influx in erythrocyte suspensions. This effect of adrenaline was inhibited by amiloride or EIPA, indicating that adrenaline stimulated NHE 1. Phorbol myristicate ester (PMA), a protein kinase C (PKC) stimulator, increased the activity of NHE 1 whereas calphostin C, a PKC inhibitor, partially inhibited NHE 1 activation induced by adrenaline. The effect of adrenaline to NHE 1 was counteracted by prazocin and by propranolol as well indicating the involvement of both alpha and beta 2 adrenergic receptors. The effect of adrenaline on erythrocyte NHE 1 activity was significantly more profound in obese compared to normal subjects. These data indicate that adrenaline induces an increase of pHi and Na(+) uptake of human erythrocytes through stimulation of NHE 1 activity. The significantly more profound stimulation of NHE 1 activity by adrenaline in obese as compared to normal subjects is discussed.

The effect of leptin on Na(+)-H(+) antiport (NHE 1) activity of obese and normal subjects erythrocytes.

Obesity is currently considered as a chronic metabolic disease, associated with a high risk of cardiovascular complications. Leptin, an adipocyte-derived hormone has a variety target cells influencing a wide range of processes. Possible counteractions of hyperleptinaemia are currently investigated. The Na(+)-H(+) exchanger (NHE 1) is involved in multiple cellular functions and its activation has been related to hypertension and obesity. NHE 1 is present on erythrocytes and can be stimulated by various hormones. Erythrocytes have on their surface a variety of receptors with mostly unknown function. In the present paper, the effect of leptin on erythrocytes NHE 1 activity has been investigated. For this reason, the intracellular pH and sodium influxes were measured before and after addition of leptin in erythrocyte suspensions from normal and obese individuals. Amiloride, a specific NHE 1 inhibitor, and staurosporine a protein kinase C inhibitor were used to inhibit erythrocyte NHE 1. For the binding study leptin was labeled with fluorescein isothiocyanate (FITC) and the binding on erythrocytes was estimated by Scatchard analysis. NHE 1 activity increased in the presence of leptin but significantly less in the obese than in the control group. Furthermore the concentrations of leptin binding sites on the surface of erythrocytes were lower in erythrocytes drawn from obese individuals than in erythrocytes drawn from normal subjects. Since NHE 1 activity has been associated with insulin resistance and hypertension, the activation of this antiport by leptin may represent a link between adipose tissue hypertrophy and cardiovascular complications of obesity.

NHE-1: a molecular target for signalling and cell matrix interactions.

The activation of sodium/hydrogen exchanger (NHE) is associated with a variety of cell functions like cell adhesion, migration, proliferation, and apoptosis. Since its discovery, 9 NHE isoforms have been identified, but the most widely spread and the most important for the cellular functions is NHE-1. This ubiquitously expressed sodium/hydrogen exchanger (NHE-1) plays a central housekeeping role in all cells regulating cell volume and internal pH (pHi). At physiological pHi, NHE-1 is essentially inactive but it is extremely sensitive to pHi changes, being rapidly activated by small intracellular hydrogen concentration increases. NHE-1 activity can be stimulated via a series of cell surface receptors, including tyrosine kinase, G-protein-coupled, and integrin receptors. These signals converge, regulating the affinity of the internal hydrogen-binding site. NHE-1 also is a plasma membrane-anchoring protein for the cytoskeleton. Cytoskeleton anchoring of NHE-1 is important for cell adhesion to extracellular matrix proteins and cell migration. Moreover, NHE-1 plays the role of a “scaffold” for the building of various intracellular signaling molecule clusters.

Effect of endothelin on sodium/hydrogen exchanger activity of human monocytes and atherosclerosis-related functions.

Abstract:  The objective of this article is to investigate the influence of endothelin‐1 (ET‐1) on human monocyte Na+/H+ exchanger (NHE) activity and on the atherosclerosis‐related monocyte functions. ET‐1 caused an increase in pHi and in 22Na influx of monocytes. A reversal of ET‐1 effect on pHi was observed in the presence of the NHE1 inhibitor, cariporide. In addition, the activation of NHE1 by ET‐1 was mediated via protein kinase C (PKC), mitogen‐activated protein kinase (MAPK), phosphatidylinositol 3‐kinase (PI3K), and NADPH oxidase. Also, a link between ET‐1 and nitric oxide (NO) was observed. Furthermore, after ET‐1 treatment, an increase of the adhesive capacity, the migration ability on laminin and CD36 expression of monocytes, was observed; using cariporide this increase was abolished. Our results showed that ET‐1 induces a signaling pathway with the involvement of PKC, MAPK, PI3K, and NADPH oxidase where NHE1 plays a key role. ET‐1 also plays a significant role in atherosclerosis‐related functions of human monocytes, via NHE1 activation.

Signaling Components Involved in Leptin-Induced Amplification of the Atherosclerosis-Related Properties of Human Monocytes

Background/Aims: Leptin, a 16-kDa cytokine that is released mainly by the adipose tissue, is known to affect a wide assortment of processes, ranging from energy homeostasis to angiogenesis and the immune response. In the present study, the effect of leptin on atherosclerosis-related properties of human monocytes was investigated. Methods: Monocytes were isolated from whole blood obtained from healthy donors who had normal body mass index values. Pharmacological inhibition of specific signaling proteins was implemented. Fluorescence spectrometry and immunofluorescence techniques, as well as ELISA methods, were utilized. Leptin dose response curves were determined for each type of experiment. Results: Leptin (160 ng/ml) was found to augment monocyte adhesion to laminin-1 and its migration through this glycoprotein, which is one of the main components of the extracellular matrix. Additionally, leptin increased CD36-receptor surface expression, as well as moderately oxidized low-density lipoprotein (oxLDL3) uptake levels. Conclusion: Leptin amplifies the pro-atheromatic properties of human monocytes through a complex signaling net which involves the Na+/H+ exchanger isoform-1, the actin cytoskeleton, phosphoinositide 3-kinase, certain conventional isoforms of protein kinase C and NADPH oxidase.