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Alcohol downregulates hepcidin expression in the liver leading to an increase in intestinal iron transport and liver iron storage. We have previously demonstrated that alcohol-mediated oxidative stress is involved in the inhibition of hepcidin transcription by alcohol in vivo. Kupffer cells and TNF-[alpha] play a key role in alcohol-induced liver injury. The aim of this study was to define their involvement in the regulation of hepcidin expression by alcohol. Kupffer cells were inactivated or depleted by employing gadolinium chloride and liposomes containing clodronate, respectively. Rats pair fed with the alcohol-Lieber-DeCarli diet for 6 wk and mice fed with 20% ethanol in the drinking water for 1 wk were used as experimental models. Interestingly, alcohol downregulated hepcidin expression in the livers of rats and mice independent of gadolinium chloride or clodronate treatment. One week of alcohol treatment was sufficient to induce a significant increase in TNF-[alpha] levels and phosphorylation of NF-[kappa]B subunit p65. The neutralization of TNF-[alpha] by specific antibodies inhibited p65 phosphorylation. However, neither the neutralization of TNF-[alpha] nor the lack of TNF-[alpha] receptor expression reversed alcohol-induced suppression of liver hepcidin expression. The level of alcohol-induced ROS in the liver was also undiminished following Kupffer cell inactivation or depletion. Our results demonstrate that alcohol-induced Kupffer cell activation and TNF-[alpha] signaling are not involved in the suppression of liver hepcidin expression by alcohol-mediated oxidative stress in vivo. Therefore, these findings suggest that alcohol acts within hepatocytes to suppress hepcidin expression and thereby influences iron homeostasis. alcoholic liver disease; iron; liver injury; oxidative stress

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [[Ca.sup.2+].sub.i] was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3-10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [[Ca.sup.2+].sub.i] in secretin- or dibulyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [[Ca.sup.2+].sub.i] in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3-10 mM concentrations significantly reduced fluid secretion and decreased [[Ca.sup.2+].sub.i] in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [[Ca.sup.2+].sub.i], response to ACh as well as the [[Ca.sup.2+].sub.i] elevation induced by thapsigargin, suggesting that they inhibit [Ca.sup.2+] influx. Removal of extracellular [Ca.sup.2+] reduced [[Ca.sup.2+].sub.i] in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [[Ca.sup.2+].sub.i] in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane [Ca.sup.2+] channel. intracellular calcium; acetylcholine