Coenzyme Q 10 supplementation: A potential therapeutic option for the treatment of intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease characterized by pruritus, elevated serum bile acids and abnormal liver function that may be associated with severe adverse pregnancy outcomes. We previously reported that plasma coenzyme Q10 (CoQ10) is decreased in women with ICP as it is its analogue coenzyme Q9 (CoQ9) in rats with ethinyl estradiol (EE)-induced cholestasis. The aim of the present study was to evaluate the possible therapeutic role of CoQ10 in experimental hepatocellular cholestasis and to compare it with ursodeoxycholic acid (UDCA) supplementation. Bile acids, CoQ9, CoQ10, transaminases, alkaline phosphatase, retinol, α-tocopherol, ascorbic acid, thiobarbituric acid reactive substances, carbonyls, glutathione, superoxide dismutase and catalase were assessed in plasma, liver and/or hepatic mitochondria in control and cholestatic rats supplemented with CoQ10 (250 mg/kg) administered alone or combined with UDCA (25 mg/kg). CoQ10 supplementation prevented bile flow decline (P < 0.05) and the increase in serum alkaline phosphatase and bile acids, particularly lithocholic acid (P < 0.05) in cholestatic rats. Furthermore, it also improved oxidative stress parameters in the liver, increased both CoQ10 and CoQ9 plasma levels and partially prevented the fall in α-tocopherol (P < 0.05). UDCA also prevented cholestasis, but it was less efficient than CoQ10 to improve the liver redox environment. Combined administration of CoQ10 and UDCA resulted in additive effects. In conclusion, present findings show that CoQ10 supplementation attenuated EE-induced cholestasis by promoting a favorable redox environment in the liver, and further suggest that it may represent an alternative therapeutic option for ICP.

The exocrine pancreas is an extracardiac source of atrial natriuretic peptide.

Previous studies have shown that atrial natriuretic peptide (ANP) regulates exocrine pancreatic function in health and disease. As extracardiac sources of ANP have been identified and ANP-like immunoreactivity has been reported in the exocrine pancreas, in the present work we sought to establish whether ANP was produced in the rat exocrine pancreas and if conditions like fasting/feeding or acute pancreatitis were reflected on ANP expression. By using RT-PCR, immunoblotting, and immunofluorescence microscopy assays, it was found that both mRNA and protein ANP were present in the acinar cells of the exocrine pancreas. The amount of ANP in the pancreas was lower in than the atrium but similar to other tissues like the kidney and liver. Immunogold labeling electron microscopy studies revealed that ANP was localized in zymogen granules and the endoplasmic reticulum suggesting local synthesis and package into granules. ANP protein expression was significantly increased not only in fasting but also in acute pancreatitis, the latter probably related to impaired secretion. Natriuretic peptide receptor type C which mediates ANP biological effects in the exocrine pancreas was also present in acinar cells and its expression did not change with either fasting or acute pancreatitis. Present findings show that the exocrine pancreas is a relatively important extracardiac source of ANP and further support previous studies strongly suggesting the active role of the peptide in pancreatic physiology and pathophysiology.

Coenzyme Q in pregnant women and rats with intrahepatic cholestasis.

BACKGROUND & AIMS: Intrahepatic cholestasis of pregnancy is a high-risk liver disease given the eventual deleterious consequences that may occur in the foetus. It is accepted that the abnormal accumulation of hydrophobic bile acids in maternal serum are responsible for the disease development. Hydrophobic bile acids induce oxidative stress and apoptosis leading to the damage of the hepatic parenchyma and eventually extrahepatic tissues. As coenzyme Q (CoQ) is considered an early marker of oxidative stress in this study, we sought to assess CoQ levels, bile acid profile and oxidative stress status in intrahepatic cholestasis. METHODS: CoQ, vitamin E and malondialdehyde were measured in plasma and/or tissues by HPLC-UV method whereas serum bile acids by capillary electrophoresis in rats with ethinyl estradiol-induced cholestasis and women with pregnancy cholestasis. RESULTS: CoQ and vitamin E plasma levels were diminished in both rats and women with intrahepatic cholestasis. Furthermore, reduced CoQ was also found in muscle and brain of cholestatic rats but no changes were observed in heart or liver. In addition, a positive correlation between CoQ and ursodeoxycholic/lithocholic acid ratio was found in intrahepatic cholestasis suggesting that increased plasma lithocholic acid may be intimately related to CoQ depletion in blood and tissues. CONCLUSION: Significant CoQ and vitamin E depletion occur in both animals and humans with intrahepatic cholestasis likely as the result of increased hydrophobic bile acids known to produce significant oxidative stress. Present findings further suggest that antioxidant supplementation complementary to traditional treatment may improve cholestasis outcome.

Endothelin-1 and -3 induce choleresis in the rat through ETB receptors coupled to nitric oxide and vagovagal reflexes.

We have reported previously that centrally applied ET (endothelin)-1 and ET-3 induce either choleresis or cholestasis depending on the dose. In the present study, we sought to establish the role of these endothelins in the short-term peripheral regulation of bile secretion in the rat. Intravenously infused endothelins induced significant choleresis in a dose-dependent fashion, ET-1 being more potent than ET-3. Endothelins (with the exception of a higher dose of ET-1) did not affect BP (blood pressure), portal venous pressure or portal blood flow. ET-1 and ET-3 augmented the biliary excretion of bile salts, glutathione and electrolytes, suggesting enhanced bile acid-dependent and -independent bile flows. ET-induced choleresis was mediated by ET(B) receptors coupled to NO and inhibited by truncal vagotomy, atropine administration and capsaicin perivagal application, supporting the participation of vagovagal reflexes. RT (reverse transcription)-PCR and Western blot analysis revealed ETA and ET(B) receptor expression in the vagus nerve. Endothelins, through ET(B) receptors, augmented the hepatocyte plasma membrane expression of Ntcp (Na⁺/taurocholate co-transporting polypeptide; Slc10a1), Bsep (bile-salt export pump; Abcb11), Mrp2 (multidrug resistance protein-2; Abcc2) and Aqp8 (aquaporin 8). Endothelins also increased the mRNAs of these transporters. ET-1 and ET-3 induced choleresis mediated by ET(B) receptors coupled to NO release and vagovagal reflexes without involving haemodynamic changes. Endothelin-induced choleresis seems to be caused by increased plasma membrane translocation and transcriptional expression of key bile transporters. These findings indicate that endothelins are able to elicit haemodynamic-independent biological effects in the liver and suggest that these peptides may play a beneficial role in pathophysiological situations where bile secretion is impaired.

Atrial natriuretic factor stimulates efflux of cAMP in rat exocrine pancreas via multidrug resistance-associated proteins.

BACKGROUND & AIMS: Atrial natriuretic factor (ANF) prevents increases in intracellular levels of cAMP that are induced by secretin in the exocrine pancreas. We investigated the contribution of cyclic adenosine monophosphate (cAMP) efflux to ANF inhibition of secretin signaling. METHODS: Intracellular and extracellular cAMP were measured by radio-binding assays in isolated pancreatic acini exposed to secretin and other secretagogues, alone or with ANF. Levels of messenger RNA for multidrug resistance-associated protein (MRP)4, MRP5, and MRP8 were measured by real-time polymerase chain reaction. MRP4 was knocked down in AR42J cells by small interfering RNA. In vivo studies were performed in rats. RESULTS: Pancreatic secretagogues increased levels of intracellular cAMP, but only secretin and vasoactive intestinal peptide promoted cAMP efflux; efflux was increased by ANF, through signaling via natriuretic peptide receptor-C and phospholipase C-protein kinase C. In time-course studies with active phosphodiesterases, levels of intracellular and extracellular cAMP increased earlier after the addition of secretin and ANF (1 min) than after the addition of secretin alone (3 min). Similar kinetic patterns occurred with a phosphodiesterase inhibitor. A probenecid-sensitive transporter mediated cAMP egression. The main cAMP transporter, MRP4, was expressed in AR42J cells and pancreas. cAMP egression occurred in AR42J cells exposed to secretin, but this response was reduced in cells that expressed MRP4 small interfering RNA. In rats, levels of cAMP in plasma and pancreatic juice increased after infusion with secretin alone or secretin plus ANF. CONCLUSIONS: ANF signals via natriuretic peptide receptor-C coupled to the phospholipase C-protein kinase C pathway to increase secretin-induced efflux of cAMP, probably through MPR-4. Cyclic AMP extrusion might be a mechanism, in addition to phosphodiesterase action, to regulate intracellular cAMP levels in pancreatic acinar cells.

Endothelins participate in the central and peripheral regulation of submandibular gland secretion in the rat.

We previously reported that endothelins (ETs) are involved in the rat central and peripheral regulation of bile secretion. In this study we sought to establish whether ET-1 and ET-3 modulated submandibular gland secretion when locally or centrally applied. Animals were prepared with gland duct cannulation to collect saliva samples and jugular cannulation to administer sialogogues. ETs were given either into the submandibular gland or brain lateral ventricle. Intraglandularly administered ETs failed to elicit salivation per se. However, ET-1, but not ET-3, potentiated both cholinergic- and adrenergic-evoked salivation through ET(A) receptors. ET-1 decreased cAMP content but increased phosphoinositide hydrolysis, whereas ET-3 attenuated both intracellular pathways. The expression of ET(A) and ET(B) receptor mRNAs as well as that of ETs was revealed in the submandibular gland by RT-PCR. Immunohistochemical studies showed that ET(A) receptor staining was localized around the interlobular ducts and acini, compatible with the myoepithelial cells' location, whereas ET(B) receptor staining was restricted to small blood vessels. When applied to the brain, both ETs induced no salivation but enhanced cholinergic- and adrenergic-evoked salivary secretion through parasympathetic pathways. ET-1 response was mediated by brain ET(A) receptors, whereas that of ET-3 was presumably through nonconventional ET receptors. Present findings show that ETs are involved in the brain regulation of cholinergic- and adrenergic-stimulated submandibular gland secretion through the activation of distinct brain ET receptors and parasympathetic pathways. However, when ETs were administered into the gland, only ET-1 enhanced cholinergic and adrenergic salivation likely through myopithelial cell contraction by activating ET(A) receptors coupled to phospholipase C. The presence of ETs and ET receptors suggests the existence of an endothelinergic system in the submandibular gland.

Atrial natriuretic factor intracellular signaling in the rat submandibular gland.

We previously reported that intravenously administered atrial natriuretic factor (ANF) induced no salivation but enhanced agonist-evoked secretion in submandibular glands. The gene expression of ANF and natriuretic peptide receptors (NPR) was later reported in the glands. In the present study we sought to establish the intracellular signalling mechanisms underlying ANF modulation of salivary secretion. Fasted rats were prepared with submandibular duct and femoral cannulation. Dose-response curves to methacholine (MC) and norepinephrine (NE) were performed in the presence of cANP (4-23 amide) (selective NPR-C agonist) and ANF. Local injection of the agonist or ANF-induced no salivation, but enhanced MC and NE-evoked secretion. ANF and cANP (4-23 amide) enhanced phosphoinositide turnover being the effect abolished by U73122 (PLC inhibitor). Further ANF and cANP (4-23 amide) decreased basal cAMP content but failed to affect isoproterenol or forskolin-evoked cAMP. ANF response was inhibited by pertussis toxin and mimicked by cANP (4-23 amide) strongly supporting NPR-C activation. ANF-induced cAMP reduction was abolished by PLC and PKC inhibitors. The content of cGMP was dose dependently stimulated by ANF but not modified by cANP (4-23 amide). These findings support that ANF through NPR-C receptors coupled to PLC activation and adenylyl cyclase inhibition interacts with sialogogic agonists in the submandibular gland to potentiate salivation.

C-type natriuretic peptide stimulates pancreatic exocrine secretion in the rat: role of vagal afferent and efferent pathways.

We previously reported that C-type natriuretic peptide (CNP) increases amylase release in isolated pancreatic acini through natriuretic peptide receptor C activation and enhances pancreatic exocrine secretion via vagal pathways when applied to the brain. In the present study we sought to establish whether CNP was involved in the peripheral regulation of pancreatic secretion. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation and bile diversion into the duodenum. CNP dose-dependently enhanced pancreatic flow, chloride and protein excretion but did not modify bicarbonate output. A selective natriuretic peptide receptor C agonist enhanced pancreatic flow and mimicked CNP-evoked protein output but failed to modify chloride secretion. Truncal vagotomy, perivagal application of capsaicin and hexamethonium reduced CNP-evoked pancreatic flow and abolished chloride excretion but did not affect protein output. Furthermore, pre-treatment with atropine reduced both CNP-stimulated pancreatic flow and chloride excretion but failed to modify protein excretion. Partial muscarinic blockade of CNP-evoked chloride output suggested that mediators other than acetylcholine were involved. However, CNP response was unaltered by cholecystokinin and vasoactive intestinal peptide receptor blockade or by nitric oxide synthase inhibition. In conclusion, CNP-stimulated pancreatic flow through the activation of the natriuretic peptide receptor C and the vago-vagal reflex but it increased protein output only by natriuretic peptide receptor C activation and chloride excretion by vago-vagal reflexes. Present results suggest that CNP may play a role as a local regulator of the exocrine pancreas.

Vagally mediated cholestatic and choleretic effects of centrally applied Endothelin-1 through ETA receptors.

The role of Endothelin-1 (ET-1) in the central nervous system is not fully understood yet although several studies strongly support its neuromodulatory role. A high density of endothelin receptors is present in the dorsal vagal complex that is the major site for the regulation of the digestive function. Therefore in the present study we sought to establish the role of ET-1 in the central regulation of bile secretion in the rat. Intracerebroventricular ET-1 injection exhibited opposite behaviors on spontaneous bile secretion according to the dose administered. Lower doses of ET-1 (1 fM) increased bile flow and bicarbonate excretion whereas higher doses (1 nM) decreased bile flow and bile acid output. Both the choleretic and the cholestatic effects of ET-1 were abolished in animals pretreated with icv BQ-610 (selective ETA antagonist) but not with BQ-788 (selective ETB antagonist). In addition, truncal vagotomy but not adrenergic blockade abolished ET-1 effects on bile secretion. Brain nitric oxide was not involved in ET-1 response since L-NAME pretreatment failed to affect ET-1 actions on the liver. Portal venous pressure was increased by centrally administered ET-1 being the magnitude of the increase similar with low and high doses of the peptide. These results show that centrally applied ET-1 modified different bile flow fractions independent of hemodynamic changes. Lower doses of ET-1 increased bile acid independent flow whereas higher doses decreased bile acid dependent flow. Vagal pathways through the activation of apparently distinct ETA receptors mediated the cholestatic as well as the choleretic effects induced by ET-1. Present findings show that ET-1 participates in the central regulation of bile secretion in the rat and give further insights into the complexity of brain-liver interaction.

C-type natriuretic peptide applied to the brain enhances exocrine pancreatic secretion through a vagal pathway.

C-type natriuretic peptide (CNP) is the major natriuretic peptide in the brain and its mRNA has been reported in the central nervous system, which supports local synthesis and its role as a neuromodulator. The aim of the present work was to study the effect of centrally applied CNP on pancreatic secretion. Rats were fitted with a lateral cerebroventricular cannula one-week before secretion studies. The central administration of CNP dose-dependently enhanced pancreatic fluid and protein output. CNP response was diminished by atropine and hexamethonium, but it was abolished by vagotomy. Neither adrenergic antagonists nor the administration of (D-p-Cl-Phe(6),Leu(17))-vasoactive intestinal peptide (VIP antagonist) or N(omega) Nitro-L arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor) affected CNP response. The effect induced by CNP was mimicked by 8-Br-cGMP but not by c-ANP-(4-23) amide (selective agonist of the natriuretic peptide receptor C). Furthermore, CNP interacted with cholecystokinin (CCK) and secretin in the brain to modify pancreatic secretion. Present findings show that centrally applied CNP enhanced pancreatic secretion through a vagal pathway and suggest that CNP response is mediated by the activation of natriuretic peptide guanylyl cyclase coupled receptors in the brain.

Endothelin-3 applied to the brain evokes opposite effects on bile secretion mediated by a central nitric oxide pathway.

We sought to establish Endothelin (ET-3) role in the central regulation of bile secretion in the rat. The intracerebroventricular (icv) injection of ET-3 evoked a cholestatic or a choleretic effect depending on the administered dose. Lower doses increased bile flow and bicarbonate excretion, whereas higher doses decreased bile flow and bile acid output. ET-3 effects were dependent on brain nitric oxide and independent of the autonomic nervous system or hemodynamic variations. A selective ETB antagonist abolished the cholestatic effect, whereas the choleretic effect was totally inhibited by either ETA or ETB selective blockade. These results show that ET-3 applied to the brain modified through a nitric oxide pathway distinct bile flow fractions depending on the administered dose and give further insights into the complexity of brain-liver interaction.