Zinc inhibits calcium-mediated and nitric oxide-mediated ion secretion in human enterocytes

Zn2+ is effective in the treatment of acute diarrhea, but its mechanisms are not completely understood. We previously demonstrated that Zn2+ inhibits the secretory effect of cyclic adenosine monophosphate but not of cyclic guanosine monophosphate in human enterocytes. The aim of the present study was to investigate whether Zn2+ inhibits intestinal ion secretion mediated by the Ca2+ or nitric oxide pathways. To investigate ion transport we evaluated the effect of Zn2+ (35 μM) on electrical parameters of human intestinal epithelial cell monolayers (Caco2 cells) mounted in Ussing chambers and exposed to ligands that selectively increased intracellular Ca2+ (carbachol 10− 6 M) or nitric oxide (interferon-γ 300 UI/ml) concentrations. We also measured intracellular Ca2+ and nitric oxide concentrations. Zn2+ significantly reduced ion secretion elicited by carbachol (− 87%) or by interferon-γ (− 100%), and inhibited the increase of intracellular Ca2+ and nitric oxide concentrations. These data indicate that Zn2+ inhibits ion secretion elicited by Ca2+ and nitric oxide by directly interacting with the enterocyte. They also suggest that Zn2+ interferes with three of the four main intracellular pathways of intestinal ion secretion that are involved in acute diarrhe

Pure uptake blockers of dopamine can reduce prolactin secretion: studies with diclofensine

The effects of diclofensine, a pure dopamine (DA) uptake inhibitor on 1) 3H-DA uptake in rat arcuate-periventricular nucleus-median eminence synaptosomes, 2) basal and K+-evoked endogenous DA release from tuberoinfundibular dopaminergic (TIDA) neurons and 3) in vivo prolactin (PRL) secretion were studied. Diclofensine, in concentrations of 0.01, 0.1 and 1 microM caused a marked decrease of 3H-DA uptake. In addition, it was unable to stimulate basal endogenous DA release which, on the contrary, was elicited by d-amphetamine in the same concentration (50 microM). On the other hand, diclofensine (50 microM) caused a 3 fold enhancement of K+-evoked DA release. Finally, the compound, when administered in vivo to male rats, significantly reduced basal serum PRL levels. The results of the present study seem to indicate that the pharmacological blockade of DA uptake in TIDA neurons is a condition sufficient to cause a reduction of PRL release

Involvement of the nitric oxide/protein kinase G pathway in polychlorinated biphenyl-induced cell death in SH-SY 5Y neuroblastoma cells

Polychlorinated biphenyls (PCB) are persistent environmental contaminants whose chronic exposure can affect nervous system development and function. The cellular and molecular mechanisms underlying neuronal damage are not yet clear. In the present study, we investigated whether nitric oxide (NO) could be involved in aroclor 1254 (A1254; a PCB mixture)-induced cytotoxicity in SH-SY5Y human neuroblastoma cells. Prolonged exposure (24 hr) to A1254 (10-100 microg/ml) caused a dose-dependent reduction of cell viability that was attenuated in the presence of a calcium entry blocker, gadolinum (Gd(3+)) at 10 microM, a concentration able to block voltage-sensitive calcium channels. In addition, A1254 caused an increase of cytosolic calcium that was dependent on extracellular calcium, as measured by fura-2 videomicroscopy. A1254-induced calcium rise may stimulate NO production through an activation of neuronal NOS (nNOS). Indeed, the concomitant addition of the selective nNOS inhibitor N(omega)-propyl-L-arginine (NPLA) and A1254 prevented cell injury, suggesting that NO production plays a major role in A1254-evoked cell injury. Furthermore, the exposure (14 hr) to A1254 (30 microg/ml) produced an up-regulation of the expression of beta isoform of nNOS. This up-regulation was calcium dependent and was accompanied by an enhancement of NO production as demonstrated by an increase of nitrite formation. Moreover, A1254-induced cell injury was prevented when KT 5823, a selective cGMP/PKG inhibitor, was added concomitantly to 30 microg/ml A1254. These results suggest that PCB-induced cell death in neuroblastoma cells is mediated by an activation of the cGMP/PKG pathway triggered by NO production

p38/Sp1/Sp4/HDAC4/BDNF axis is a novel molecular pathway of the neurotoxic effect of the methylmercury

The molecular pathways involved in methylmercury (MeHg)-induced neurotoxicity are not fully understood. Since pan-Histone deacetylases (HDACs) inhibition has been found to revert the neurodetrimental effect of MeHg, it appeared of interest to investigate whether the pattern of HDACs isoform protein expression is modified during MeHg-induced neurotoxicity and the transcriptional/transductional mechanisms involved. SH-SY5Y neuroblastoma cells treated with MeHg 1 μM for 12 and 24 h showed a significant increase of HDAC4 protein and gene expression, whereas the HDACs isoforms 1-3, 5, and 6 were unmodified. Furthermore, MeHg-induced HDAC4 increase was reverted when cells were transfected with siRNAs against specificity protein 1 (Sp1) and Sp4, that were both increased during MeHg exposure. Next we studied the role of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) in MeHg-induced increase of Sp1, Sp4, and HDAC4 expression. As shown by Western Blot analysis MeHg exposure increased the phosphorylation of p38, but not of ERK and JNK. Notably, when p38 was pharmacologically blocked, MeHg-induced Sp1, Sp4 protein expression, and HDAC4 protein and gene expression was reverted. In addition, MeHg exposure increased the binding of HDAC4 to the promoter IV of the Brain-derived neurotrophic factor (BDNF) gene, determining its mRNA reduction, that was significantly counteracted by HDAC4 knocking down. Furthermore, rat cortical neurons exposed to MeHg (1 μM/24 h) showed an increased phosphorylation of p38, in parallel with an up-regulation of Sp1, Sp4, and HDAC4 and a down-regulation of BDNF proteins. Importantly, transfection of siRNAs against p38, Sp1, Sp4, and HDAC4 or transfection of vector overexpressing BDNF significantly blocked MeHg-induced cell death in cortical neurons. All these results suggest that p38/Sp1-Sp4/HDAC4/BDNF may represent a new pathway involved in MeHg-induced neurotoxicity

Zn(2+) slows down Ca(V)3.3 gating kinetics: implications for thalamocortical activity

We employed whole cell patch-clamp recordings to establish the effect of Zn(2+) on the gating the brain specific, T-type channel isoform Ca(V)3.3 expressed in HEK-293 cells. Zn(2+) (300 microM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca(2+) currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn(2+) slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn(2+) also decreased whole cell Ca(2+) permeability to 45% of control values. In the presence of Zn(2+), Ca(2+) currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn(2+) on T-type channels (while leaving the kinetic parameters of voltage-gated Na(+) and K(+) unchanged) revealed that Zn(2+) increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn(2+) decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 microM). These data demonstrate that Zn(2+) modulates Ca(V)3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn(2+) may have a role in controlling thalamocortical oscillations