ucn-1028-c and ethylisopropylamiloride

The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in *O2- production, with Cd to be more potent (216 +/- 15%) than Zn (150 +/- 9.5%), in relation to control value (100%). The metals effect was reversed after incubation with the amiloride analogue, EIPA, a selective Na+/H+ exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on *O2- production was mediated via the interaction of metal ions with alpha1- and beta-adrenergic receptors, as shown after incubation with their respective agonists and antagonists. In addition, both metals caused an increase in intracellular pH (pHi) of gill cells. EIPA together with either metal significantly reduced the effect of each metal treatment on pHi. Incubation of gill cells with the oxidants rotenone, antimycin A and pyruvate caused a significant increase in pHi (delta pHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi (delta pHi -0.090) was measured, indicating that change in reactive oxygen species (ROS) production by heavy metals affects NHE activity. When rosiglitazone was incubated together with either heavy metal a decrease in O2- production was observed. Our results show a key role of NHE in the signal transduction pathway induced by Zn and Cd in gill cells, with the involvement of ROS, PKC, adrenergic and PPAR-gamma receptors. In addition, differences between the two metals concerning NHE activation, O2- production and interaction with adrenergic receptors were observed.

Topics: Acetylcysteine; Amiloride; Animals; Antimycin A; Antioxidants; Cadmium; Cadmium Poisoning; Cell Survival; Gills; Hydrogen-Ion Concentration; Mytilus; Naphthalenes; Oxidants; PPAR gamma; Protein Kinase Inhibitors; Pyruvic Acid; Reactive Oxygen Species; Receptors, Adrenergic; Rosiglitazone; Rotenone; Sodium-Hydrogen Exchangers; Thiazolidinediones; Water Pollutants, Chemical; Zinc

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 micro mol 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.

Topics: Amiloride; Animals; Bivalvia; Cadmium; Digestive System; Epinephrine; Greece; Hydrogen-Ion Concentration; Isoproterenol; Naphthalenes; Phenylephrine; Phorbol Esters; Prazosin; Propranolol; Protein Kinase C; Pyruvate Kinase; Signal Transduction; Sodium-Hydrogen Exchangers; Staurosporine

To evaluate further the signal transduction mechanisms involved in the short-term modulation of Na-K-ATPase activity in the mammalian kidney, we examined the role of phospholipase C-protein kinase C (PLC-PKC) pathway and of various eicosanoids in this process, using microdissected rat proximal convoluted tubules. Dopamine (DA) and parathyroid hormone (either synthetic PTH1-34 or PTH3-34) inhibited Na-K-ATPase activity in dose-dependent manner; this effect was reproduced by PKC530-558 fragment and blocked by the specific PKC inhibitor calphostin C, as well as by the PLC inhibitors neomycin and U-73122. Pump inhibition by DA, PTH, or arachidonic acid, and by PKC activators phorbol dibutyrate (PDBu) or dioctanoyl glycerol (DiC8) was abolished by ethoxyresorufin, an inhibitor of the cytochrome P450-dependent monooxygenase pathway, but was unaffected by indomethacin or nordihydroguaiaretic acid, inhibitors of the cyclooxygenase and lipoxygenase pathways of the arachidonic acid cascade, respectively. Furthermore, each of the three monooxygenase products tested (20-HETE, 12(R)-HETE, or 11,12-DHT) caused a dose-dependent inhibition of the pump. The effect of DA, PTH, PDBu or DiC8, as well as that of 20-HETE was not altered when sodium entry was blocked with the amiloride analog ethylisopropyl amiloride or increased with nystatin. We conclude that short-term regulation of proximal tubule Na-K-ATPase activity by dopamine and parathyroid hormone occurs via the PLC-PKC signal transduction pathway and is mediated by cytochrome P450-dependent monooxygenase products of arachidonic acid metabolism, which may interact with the pump rather than alter sodium access to it.

Topics: Amiloride; Animals; Dopamine; Eicosanoids; Enzyme Inhibitors; Estrenes; Homeostasis; Indomethacin; Kidney Tubules, Proximal; Kinetics; Male; Masoprocol; Models, Biological; Naphthalenes; Neomycin; Nystatin; Oxazines; Parathyroid Hormone; Peptide Fragments; Protein Kinase C; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Teriparatide; Type C Phospholipases