Long-term effect of Toll-like receptor-2, -4, -5, -7 and NOD2 stimulation on Na+,K+-ATPase activity and expression in intestinal epithelial cells.

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and ß1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.

Effect of Toll-like receptor-2, -4, -5, -7, and NOD2 stimulation on potassium channel conductance in intestinal epithelial cells.

Disproportionate activation of pattern recognition receptors plays a role in inflammatory bowel disease (IBD) pathophysiology. Diarrhea is a hallmark symptom of IBD, resulting at least in part from an electrolyte imbalance that may be caused by changes in potassium channel activity. We evaluated the impact of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) stimulation on potassium conductance of the basolateral membrane in human intestinal epithelial cells (IECs) and the role of potassium channels through electrophysiological assays under short-circuit current in Ussing chambers. TLRs and NOD2 were stimulated using specific agonists, and potassium channels were selectively blocked using triarylmethane-34 (TRAM-34), adenylyl-imidodiphosphate (AMP-PNP), and BaCl2. Potassium conductance of the basolateral membrane decreased upon activation of TLR2, TLR4, and TLR7 in T84 cells (means ± SE, -11.2 ± 4.5, -40.4 ± 7.2, and -19.4 ± 5.9, respectively) and in Caco-2 cells (-13.1 ± 5.7, -55.7 ± 7.4, and -29.1 ± 7.2, respectively). In contrast, activation of TLR5 and NOD2 increased basolateral potassium conductance, both in T84 cells (18.0 ± 4.1 and 18.4 ± 2.8, respectively) and in Caco-2 cells (21.2 ± 8.4 and 16.0 ± 3.6, respectively). TRAM-34 and AMP-PNP induced a decrease in basolateral potassium conductance upon TLR4 stimulation in both cell lines. Both KCa3.1- and Kir6-channels appear to be important mediators of this effect in IECs and could be potential targets for therapeutic agent development.NEW & NOTEWORTHY This study highlights that PRRs stimulation directly influences K+-channel conductance in IECs. TLR-2, -4, -7 stimulation decreased K+ conductance, whereas TLR5 and NOD2 stimulation had the opposite effect, leading to an increase of it instead. This study reports for the first time that KCa3.1- and Kir6-channels play a role in K+ transport pathways triggered by TLR4 stimulation. These findings suggest that KCa3.1- and Kir6-channels modulation may be a potential target for new therapeutic agents in IBD.

Salt-inducible kinases: new players in pulmonary arterial hypertension?

Salt-inducible kinases (SIKs) are serine/threonine kinases belonging to the AMP-activated protein kinase (AMPK) family. Accumulating evidence indicates that SIKs phosphorylate multiple targets, including histone deacetylases (HDACs) and cAMP response element-binding protein (CREB)-regulated transcriptional coactivators (CRTCs), to coordinate signaling pathways implicated in metabolism, cell growth, proliferation, apoptosis, and inflammation. These pathways downstream of SIKs are altered not only in pathologies like cancer, systemic hypertension, and inflammatory diseases, but also in pulmonary arterial hypertension (PAH), a multifactorial disease characterized by pulmonary vasoconstriction, inflammation and remodeling of pulmonary arteries owing to endothelial dysfunction and aberrant proliferation of smooth muscle cells (SMCs). In this opinion article, we present evidence of SIKs as modulators of key signaling pathways involved in PAH pathophysiology and discuss the potential of SIKs as therapeutic targets for PAH, emphasizing the need for deeper molecular insights on PAH.

The role of salt-inducible kinases on the modulation of renal and intestinal Na+,K+-ATPase activity during short- and long-term high-salt intake.

Type 1 salt-inducible kinases (SIK1) has been shown to act as a mediator during the cellular adaptation to variations in intracellular sodium in a variety of cell types. Type 2 SIK (SIK2) modulates various biological functions and acts as a signal transmitter in various pathways. To evaluate the role of both SIK isoforms in renal and intestinal Na+,K+-ATPase (NKA) activity, we made use of constitutive sik1-/- (SIK1-KO), sik2-/- (SIK2-KO), double sik1-/-sik2-/- (double SIK1*2-KO) knockout and wild-type (WT) mice challenged to a standard (0.3% NaCl) or chronic high-salt (HS, 8% NaCl) diet intake for 48 h or 12 weeks. Long-term HS intake in WT was accompanied by 2-fold increase in jejunal NKA activity and slight (~30% reduction) decreases in NKA in the ileum and cecum; none of these changes was accompanied by changes in the expression of α1-NKA. The ablation of SIK1 and SIK2 prevented the marked increase in jejunal NKA activity following the long-term HS intake. The ablation of SIK1 and SIK2 in mice on a long-term HS intake impacted differently in the ileum and cecum. The most interesting finding is that in SIK2-KO mice marked reductions in NKA activity were observed in the ileum and cecum when compared to WT mice, both on normal and long-term HS intake. In summary, SIK1 or SIK2 ablation on chronic high-salt intake is accompanied by modulation of NKA along the intestinal tract, which differ from those after an acute high-salt intake, and this may represent an absorptive compensatory mechanism to keep electrolyte homeostasis.

Repurposing nitrocatechols: 5-Nitro-α-cyanocarboxamide derivatives of caffeic acid and caffeic acid phenethyl ester effectively inhibit aggregation of tau-derived hexapeptide AcPHF6.

Polyphenols like caffeic acid and its phenethyl ester have been associated with potent anti-aggregating activity. Accordingly, we screened a library of polyphenols and synthetic derivatives thereof for their capacity to inhibit tau-aggregation using a thioflavin T-based fluorescence method. Our results show that the nitrocatechol scaffold is required for a significant anti-aggregating activity, which is enhanced by introducing bulky substituents at the side chain. A remarkable increase in activity was observed for α-cyanocarboxamide derivatives 26-27. Molecular docking studies showed that the amide bond provides superior conformational stability in the steric zipper assembly of tau, which drives the increase in activity. We also found that derivatives 24-27 were potent chelators of copper(II) - a property of pharmacological significance in abnormal protein aggregation. These small molecules can provide promising leads to develop new drugs for tauopathies and AD. These findings open a new window on the repurposing of nitrocatechols beyond their established role as catechol-O-methyltransferase inhibitors.

Pharmacodynamic evaluation of novel Catechol-O-methyltransferase inhibitors.

Currently, peripheral COMT inhibitors have an important role in the treatment of Parkinson's disease, and central COMT inhibitors have a potential role in the treatment of various neuropsychiatric disorders, such as attention deficit hyperactivity disorder. Adverse reactions, low bioavailability and short elimination half-lives have prompted the development of new selective COMT inhibitors. The objective of this study was to evaluate the pharmacodynamic properties of novel tight-binding COMT inhibitors (NC, NE, NDE, NCAPE, CNCAFBn, CNCAPE, NCAFBn, CNCAPA, CNCABA and CNCAHA) and compared to standard inhibitors tolcapone and entacapone. The activity of soluble (S) and membrane bound (MB) COMT from rat liver and brain was assessed in the presence of varying concentrations of each inhibitor. NE and NC behaved most potently against liver S-COMT, and CNCAPE was the most potent inhibitor against brain MB-COMT. The cytotoxicity of tolcapone and CNCAPE in human neuroblastoma SK-N-SH cells and human liver adenocarcinoma SK-HEP-1 cells was also assessed. At lower concentrations, CNCAPE did not reduce cell viability, suggesting that CNCAPE may have a potential therapeutic role as a centrally active COMT inhibitor.

Safety, Tolerability and Efficacy of Eslicarbazepine Acetate as Adjunctive Therapy in Patients Aged ≥ 65 Years with Focal Seizures.

BACKGROUND: The incidence of epilepsy is high within the first few years of life, stabilizes over the second through fifth decades, and then rises again. Treatment of elderly patients with antiepileptic drugs (AEDs) is complicated by increased sensitivity to drug effects, altered pharmacokinetics and an increased risk for drug interactions due to polytherapy. On the other hand, the safety and efficacy data of AEDs attained during clinical development programmes are relatively limited for this age group. OBJECTIVE: The aim of this study was to evaluate the safety, tolerability and efficacy of eslicarbazepine acetate (ESL) as adjunctive therapy in patients aged ≥ 65 years with focal-onset seizures (FOS). METHODS: This was an international, multicentre, open-label, non-controlled, single-arm, post-European approval commitment study with flexible doses of ESL between 400 and 1200 mg/day. Seventy-two elderly patients with at least two FOS in the prior 4 weeks, and treated with one or two AEDs, were enrolled. The study consisted of an 8-week baseline, followed by a 26-week treatment period during which the investigator was allowed to up- or down-titrate the ESL dose, and a 4-week follow-up period. Safety and tolerability were assessed as well as mental sedation, cognitive mental state and suicidal ideation. Efficacy was assessed based on patient diaries regarding the absolute and relative changes in seizure frequency, change in intellectual impairment and quality of life. RESULTS: Overall, 47 (65.3%) patients experienced 152 treatment-emergent adverse events (TEAEs). The most frequent were dizziness (12.5%), somnolence (9.7%), fatigue, convulsion and hyponatraemia (8.3% each). All patients that experienced hyponatraemia (6/72) recovered without sequelae. Three patients died during the study (due to cardiac failure, glioblastoma multiforme and ischaemic stroke, all considered unrelated to ESL). Overall, 16 (22.2%) patients discontinued prematurely due to TEAEs. The incidences of clinically significant findings were low for vital signs, ECG, physical and neurological examinations. No TEAEs of hypothyroidism were reported; however, 24 (33.3%) patients presented post-baseline shifts from normal to decreased free T4 levels (not clinically significant). ESL decreased standardized seizure frequency from a mean of 4.8 seizures at baseline to 3.6 seizures at endpoint (p > 0.05); and mean number of days with seizures significantly decreased from 4.1 (baseline) to 2.8 at endpoint (p = 0.0408). CONCLUSION: ESL taken once daily (400-1200 mg) as adjunctive therapy in patients aged ≥ 65 years was found to be safe, well tolerated and efficacious (EudraCT number: 2009-012587-14).

In vitro assessment of the interactions of dopamine ß-hydroxylase inhibitors with human P-glycoprotein and Breast Cancer Resistance Protein.

Inhibition of the biosynthesis of noradrenaline is a currently explored strategy for the treatment of hypertension, congestive heart failure and pulmonary arterial hypertension. While some dopamine ß-hydroxylase (DBH) inhibitors cross the blood-brain barrier (BBB) and cause central as well as peripheral effects (nepicastat), others have limited access to the brain (etamicastat, zamicastat). In this context, peripheral selectivity is clinically advantageous, in order to prevent alterations of noradrenaline levels in the CNS and the occurrence of adverse central effects. A limited brain exposure results from the combination of several factors, such as a reduced passive permeability or affinity for efflux transporters, but efflux liabilities may also lead to unwanted drug-drug interactions (DDIs) in the presence of co-administered substrates or inhibitors. Thus, the purpose of the study herein presented was to explore the interaction of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), the two major efflux transporters of the BBB that hamper the entry of several drugs to the brain, with the DBH inhibitors, etamicastat, nepicastat and zamicastat. Madin-Darby canine kidney cells (MDCK II) and transfected lines with human MDR1 (MDCK-MDR1) and ABCG2 (MDCK-BCRP) genes were used as a BBB surrogate model. P-gp and BCRP substrates and/or inhibitors were identified through intracellular accumulation and bidirectional permeability assays. The obtained data revealed that zamicastat is a concentration-dependent dual P-gp and BCRP inhibitor with IC50 values of 73.8 ±â€¯7.2 µM and 17.0 ±â€¯2.7 µM, while etamicastat and nepicastat inhibited BCRP to greater extent than P-gp, with IC50 values of 47.7 ±â€¯1.8 µM and 59.2 ±â€¯9.4 µM, respectively. Additionally, etamicastat was identified as P-gp and BCRP dual substrate, as demonstrated by net flux ratios of 5.84 and 3.87 and decreased >50% by verapamil and Ko143. Conversely, nepicastat revealed to be a P-gp-only substrate, with a net flux ratio of 2.01, reduced to 0.92 in the presence of verapamil. Furthermore, nepicastat displayed a consistently higher apparent permeability (>8.49 × 10-6 cm s-1) than etamicastat (<0.58 × 10-6 cm s-1). The identification of etamicastat as a dual efflux substrate suggests that P-gp and BCRP may be partially responsible for the limited central exposure of this compound, in association with its low passive permeability. Moreover, the weak efflux inhibitory potencies of etamicastat and nepicastat revealed a low DDI risk, while the dual P-gp/BCRP inhibition of zamicastat could be studied in the future with synergically effluxed compounds, for which BBB penetration is severely impaired.

Elucidation of the Impact of P-glycoprotein and Breast Cancer Resistance Protein on the Brain Distribution of Catechol-O-Methyltransferase Inhibitors.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are clinically important efflux transporters that act cooperatively at the blood-brain barrier, limiting the entry of several drugs into the central nervous system (CNS) and affecting their pharmacokinetics, therapeutic efficacy, and safety. In the present study, the interactions of catechol-O-methyltransferase (COMT) inhibitors (BIA 9-1059, BIA 9-1079, entacapone, nebicapone, opicapone, and tolcapone) with P-gp and BCRP were investigated to determine the contribution of these transporters in their access to the brain. In vitro cellular accumulation and bidirectional transport assays were conducted in Madin-Darby canine kidney (MDCK) II, MDCK-MDR1, and MDCK-BCRP cells. In vivo pharmacokinetic studies were carried out for tolcapone and BIA 9-1079 in rats, with and without elacridar, a well-known P-gp and BCRP modulator. The results suggest that BIA 9-1079, nebicapone, and tolcapone inhibit BCRP in a concentration-dependent manner. Moreover, with net flux ratios higher than 2 and decreased over 50% in the presence of verapamil or Ko143, BIA 9-1079 was identified as a P-gp substrate while BIA 9-1059, entacapone, opicapone, and nebicapone were revealed to be BCRP substrates. In vivo, brain exposure was limited for tolcapone and BIA 9-1079, although tolcapone crossed the blood-brain barrier at a greater rate and to a greater extent than BIA 9-1079. The extent of brain distribution of both compounds was significantly increased in the presence of elacridar, attesting to the involvement of efflux transporters. These findings provide relevant information and improve the understanding of the mechanisms that govern the access of these COMT inhibitors to the CNS.

The effect of PRR ligands on the membrane potential of intestinal epithelial cells.

BACKGROUND: Disproportionate signaling through intestinal epithelial pattern recognition receptors (PRR) plays a role in IBD (inflammatory bowel disease) pathophysiology. Diarrhea is a clinical trademark of IBD and altered activity of K+ channels (KC) may contribute to the low sodium absorption state. Here we sought to study the impact of PRR activation on the membrane potential of human intestinal epithelial cells and the role of KC in it. METHODS: All assays were performed in cultured HT-29 cells. KC activity was assessed by spectrofluorometry, measuring changes in cell membrane potential (MP) with the anionic fluorophore DiBAC4(3). PRRs were activated by specific ligands (MDP, LTA, MPLA, flagellin, loxoribine and ODN2216). KC modulators employed were BaCl2, pinacidil, noxiustoxin and AMP-PNP. RESULTS: Activation of NOD2, TLR5, TLR7 and TLR9 hyperpolarized the membrane (at 103ng/ml, the normalized AUC of the fluorescence intensity variation from the control were respectively (mean±SEM): -725.3±111.5; -1517.4±95.0; -857.8±61.1 and -995.6±53.6), while TLR2 and TLR4 stimulation induced membrane depolarization (1110.4±73.1 and 3890.3±342.7 at 103ng/ml, respectively). MPD effect on MP was abolished by BaCl2, partially reversed by AMP-PNP (aKATP channel inhibitor) and insensitive to noxiustoxin (a voltage-gated KC inhibitor). CONCLUSION: It was shown for the first time that PRR activation affects MP in human intestinal epithelial cells. KC appear to be important mediators in this phenomenon; in particular, KATP channels may partake in NOD2-derived effects.

Role of epithelial ion transports in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with a complex pathogenesis. Diarrhea is a highly prevalent and often debilitating symptom of IBD patients that results, at least in part, from an intestinal hydroelectrolytic imbalance. Evidence suggests that reduced electrolyte absorption is more relevant than increased secretion to this disequilibrium. This systematic review analyses and integrates the current evidence on the roles of epithelial Na(+)-K(+)-ATPase (NKA), Na(+)/H(+) exchangers (NHEs), epithelial Na(+) channels (ENaC), and K(+) channels (KC) in IBD-associated diarrhea. NKA is the key driving force of the transepithelial ionic transport and its activity is decreased in IBD. In addition, the downregulation of apical NHE and ENaC and the upregulation of apical large-conductance KC all contribute to the IBD-associated diarrhea by lowering sodium absorption and/or increasing potassium secretion.

Amine neurotransmitters, inflammation and epithelial sodium transport.

NEW FINDINGS: What is the topic of this review? The present work reviews the roles of renal and intestinal dopamine and 5-HT in the maintenance of fluid and electrolyte homeostasis. The role of inflammatory agents at the intestinal level that affect fluid and electrolyte homeostasis is also addressed. What advances does it highlight? General mechanisms of epithelial cell ion transport in the gastrointestinal tract and kidney share considerable similarities, particularly with regard to basolateral Na(+) ,K(+-) ATPase as a driving force for the movement of numerous substrates across the cell membrane. The physiological importance of the renal actions of monoamines (dopamine, noradrenaline and 5-HT) mainly depends on the sources of the amines in the kidney and on their availability to activate the amine-specific receptors. Dopamine and 5-HT are also relatively abundant in the mucosal cell layer of the intestine, and recent evidence suggests their physiological relevance in regulating electrolyte transport. The gastrointestinal tract can be an important site for the loss of water and electrolytes, in the presence of intestinal inflammation. General mechanisms of epithelial cell ion transport in the gastrointestinal tract and kidney share considerable similarities with regard to basolateral Na(+) ,K(+) -ATPase as a driving force for the movement of numerous substrates across the cell membrane. The present work reviews the roles of renal and intestinal dopamine and 5-HT in the maintenance of fluid and electrolyte homeostasis. The role of inflammatory agents at the intestinal level that affect fluid and electrolyte homeostasis is also addressed.

Short- and long-term regulation of intestinal Na+/H+ exchange by Toll-like receptors TLR4 and TLR5.

Inappropriate activation of pattern recognition receptors has been described as a potential trigger in the development of inflammatory bowel disease (IBD). In this study, we evaluated the activity and expression of Na(+)/H(+) exchanger (NHE) subtypes in T84 intestinal epithelial cells during Toll-like receptor 4 (TLR4) activation by monophosphoryl lipid A and TLR5 by flagellin. NHE activity and intracellular pH were evaluated by spectrofluorescence. Additionally, kinase activities were evaluated by ELISA, and siRNA was used to specifically inhibit adenylyl cyclase (AC). Monophosphoryl lipid A (MPLA) (0.01-50.00 µg/ml) and flagellin (10-500 ng/ml) inhibited NHE1 activity in a concentration-dependent manner (MPLA short term -25.2 ± 5.0%, long term -31.9 ± 4.0%; flagellin short term -14.9 ± 2.0%, long term -19.1 ± 2.0%). Both ligands triggered AC3, PKA, PLC, and PKC signal molecules. Long-term exposure to flagellin and MPLA induced opposite changes on NHE3 activity; flagellin increased NHE3 activity (∼10%) with overexpression of membrane protein, whereas MPLA decreased NHE3 activity (-17.3 ± 3.0%). MPLA and flagellin simultaneously had synergistic effects on NHE activity. MPLA and flagellin impaired pHi recovery after intracellular acidification. The simultaneous exposure to MPLA and flagellin induced a substantial pHi reduction (-0.55 ± 0.03 pH units). Activation of TLR4 and TLR5 exerts marked inhibition of NHE1 activity in intestinal epithelial cells. Transduction mechanisms set into motion during TLR4-mediated and long-term TLR5-mediated inhibition of NHE1 activity involve AC3, PKA, PLC, and PKC. However, short- and long-term TLR4 activation and TLR5 activation might use different signaling pathways. The physiological alterations on intestinal epithelial cells described here may be useful in the development of better IBD therapeutics.

Blood pressure decrease in spontaneously hypertensive rats folowing renal denervation or dopamine ß-hydroxylase inhibition with etamicastat.

Overactivity of the sympathetic nervous system has an important role in the development and progression of arterial hypertension. Catheter-based renal nerve ablation for the treatment of drug-resistant hypertension has recently been developed. An alternative strategy for the modulation of sympathetic nerve function is to reduce the biosynthesis of noradrenaline (NA) by inhibiting dopamine ß-hydroxylase (DßH), the enzyme that catalyzes the conversion of dopamine (DA) to NA in the sympathetic nerves. Renal denervation (RDN) surgery was performed in spontaneously hypertensive rats (SHR) to evaluate the effect of RDN on the DA and NA levels and on blood pressure over a 28-day period. The selective peripheral DßH inhibitor etamicastat (30 mg kg (-1)day(-1)) was administered to another cohort of SHR. RDN and etamicastat treatment had no effect on the renal function, as assessed by measuring the water balance response, renal function and urinary electrolyte levels. RDN significantly decreased the systolic blood pressure (SBP) and the diastolic blood pressure (DBP). A gradual return of the SBP and the DBP to the high baseline levels was observed over time. Conversely, treatment with etamicastat resulted in a significant decrease in the SBP and the DBP at all time points. On the last day of the assessment, NA levels in renal tissue were significantly decreased in both RDN and etamicastat-treated groups. In contrast, the NA levels in the left ventricle were decreased only in the etamicastat-treated group. Thus, RDN produces transitory decreases in blood pressure, whereas prolonged downregulation of sympathetic drive with the DßH inhibitor etamicastat results in a sustained decrease in the SBP and the DBP.

Carbamazepine and oxcarbazepine, but not eslicarbazepine, enhance excitatory synaptic transmission onto hippocampal CA1 pyramidal cells through an antagonist action at adenosine A1 receptors.

This study assessed the anticonvulsant and seizure generation effects of carbamazepine (CBZ), oxcarbazepine (OXC) and eslicarbazepine (S-Lic) in wild-type mice. Electrophysiological recordings were made to discriminate potential cellular and synaptic mechanisms underlying anti- and pro-epileptic actions. The anticonvulsant and pro-convulsant effects were evaluated in the MES, the 6-Hz and the Irwin tests. Whole-cell patch-clamp recordings were used to investigate the effects on fast excitatory and inhibitory synaptic transmission in hippocampal area CA1. The safety window for CBZ, OXC and eslicarbazepine (ED50 value against the MES test and the dose that produces grade 5 convulsions in all mice), was 6.3, 6.0 and 12.5, respectively. At high concentrations the three drugs reduced synaptic transmission. CBZ and OXC enhanced excitatory postsynaptic currents (EPSCs) at low, therapeutically-relevant concentrations. These effects were associated with no change in inhibitory postsynaptic currents (IPSCs) resulting in altered balance between excitation and inhibition. S-Lic had no effect on EPSC or IPSC amplitudes over the same concentration range. The CBZ mediated enhancement of EPSCs was blocked by DPCPX, a selective antagonist, and occluded by CCPA, a selective agonist of the adenosine A1 receptor. Furthermore, reduction of endogenous adenosine by application of the enzyme adenosine deaminase also abolished the CBZ- and OXC-induced increase of EPSCs, indicating that the two drugs act as antagonists at native adenosine receptors. In conclusion, CBZ and OXC possess pro-epileptic actions at clinically-relevant concentrations through the enhancement of excitatory synaptic transmission. S-Lic by comparison has no such effect on synaptic transmission, explaining its lack of seizure exacerbation.

Cardiovascular safety pharmacology profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor.

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 44.0µg/ml in HEK 293 cells. At 0.3 and 3µg/ml, etamicastat had no effects on the action potential (AP) in male dog Purkinje fibers. At 30µg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (-4%), AP duration at 60% (-14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious male dog, etamicastat (up to 20mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20mg/kg, the QTc interval was slightly prolonged (8-9% max, P<0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post-administration) were 1.4 and 3.7µg/ml at 10 and 20mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3µg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~0.2µg/ml).

Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide.

This study aimed at evaluating the effects of eslicarbazepine, carbamazepine (CBZ), oxcarbazepine (OXC) and lacosamide (LCM) on the fast and slow inactivated states of voltage-gated sodium channels (VGSC). The anti-epileptiform activity was evaluated in mouse isolated hippocampal slices. The anticonvulsant effects were evaluated in MES and the 6-Hz psychomotor tests. The whole-cell patch-clamp technique was used to investigate the effects of eslicarbazepine, CBZ, OXC and LCM on sodium channels endogenously expressed in N1E-115 mouse neuroblastoma cells. CBZ and eslicarbazepine exhibit similar concentration dependent suppression of epileptiform activity in hippocampal slices. In N1E-115 mouse neuroblastoma cells, at a concentration of 250 µM, the voltage dependence of the fast inactivation was not influenced by eslicarbazepine, whereas LCM, CBZ and OXC shifted the V0.5 value (mV) by -4.8, -12.0 and -16.6, respectively. Eslicarbazepine- and LCM-treated fast-inactivated channels recovered similarly to control conditions, whereas CBZ- and OXC-treated channels required longer pulses to recover. CBZ, eslicarbazepine and LCM shifted the voltage dependence of the slow inactivation (V0.5, mV) by -4.6, -31.2 and -53.3, respectively. For eslicarbazepine, LCM, CBZ and OXC, the affinity to the slow inactivated state was 5.9, 10.4, 1.7 and 1.8 times higher than to the channels in the resting state, respectively. In conclusion, eslicarbazepine did not share with CBZ and OXC the ability to alter fast inactivation of VGSC. Both eslicarbazepine and LCM reduce VGSC availability through enhancement of slow inactivation, but LCM demonstrated higher interaction with VGSC in the resting state and with fast inactivation gating.

Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat.

Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522) nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121) nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions.

Short- and long-term regulation of intestinal Na+/H+ exchange activity associated with TLR2 receptor activation is independent of nuclear factor-κB signaling.

Type 2 Toll-like receptors (TLR2s) are expressed in cell membranes and recognize a wide range of pathogen-associated molecular patterns derived from bacteria, such as lipoteichoic acid (LTA). The aim of this study was to evaluate the effect of TLR2 activation by LTA on the activity of type 1 Na(+)/H(+) exchanger (NHE) in T84 intestinal epithelial cells. Short-term (0.5 hour) and long-term (18 hours) TLR2 activation significantly inhibited NHE1 activity in a concentration-dependent manner (0.01-100 µg/ml; -7 ± 3 to -21 ± 3% and 3 ± 3 to -21 ± 3% of control values, respectively). S3226 [3-[2-(3-guanidino-2-methyl-3-oxopropenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride], an NHE3-selective inhibitor, did not affect the inhibitory effect on NHE activity. LTA-induced NHE inhibition did not occur in the presence ofethylisopropylamiloride (an NHE1 inhibitor). Long-term TLR2 activation decreased NHE1 affinity for Na(+) (Km= 64.98 ± 1.67 mM) compared with control (Km= 20.44 ± 0.54 mM) without changes in Vmax values. After TLR2 activation, we observed tyrosine-protein kinase (SRC) activation, phosphatidylinositol 3-kinase (PI3K) recruitment, and adenylyl cyclase (AC3) phosphorylation. The total amount of AC3 increased (23 ± 8% of control) after long-term treatment with LTA. Anti-AC3 small interfering RNA prevented LTA-induced NHE1 inhibition, similar to that observed with the AC3 inhibitor KH7 [(±)-2-(1H-benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide]. A significant increase in cAMP levels (32 ± 3% and 14 ± 2% after short- and long-term stimulation, respectively) was detected, and inhibition of protein kinase A (PKA), phospholipase C (PLC), and downregulation of protein kinase C (PKC) prevented NHE1 inhibition. Inhibition of nuclear factor-κΒ (NF-κB) failed to revert NHE1 inhibition. We concluded that activation of TLR2 reduces NHE1 activity in epithelial cells through an alternative pathway that is unrelated to NF-κB, which involves SCR, PI3K, AC3, PKA, PLC, and PKC.

Increases in intracellular sodium activate transcription and gene expression via the salt-inducible kinase 1 network in an atrial myocyte cell line.

Cardiac hypertrophy (CH) generally occurs as the result of the sustained mechanical stress caused by elevated systemic arterial blood pressure (BP). However, in animal models, elevated salt intake is associated with CH even in the absence of significant increases in BP. We hypothesize that CH is not exclusively the consequence of mechanical stress but also of other factors associated with elevated BP such as abnormal cell sodium homeostasis. We examined the effect of small increases in intracellular sodium concentration ([Na(+)](i)) on transcription factors and genes associated with CH in a cardiac cell line. Increases in [Na(+)](i) led to a time-dependent increase in the expression levels of mRNA for natriuretic peptide and myosin heavy chain genes and also increased myocyte enhancer factor (MEF)2/nuclear factor of activated T cell (NFAT) transcriptional activity. Increases in [Na(+)](i) are associated with activation of salt-inducible kinase 1 (snflk-1, SIK1), a kinase known to be critical for cardiac development. Moreover, increases in [Na(+)](i) resulted in increased SIK1 expression. Sodium did not increase MEF2/NFAT activity or gene expression in cells expressing a SIK1 that lacked kinase activity. The mechanism by which SIK1 activated MEF2 involved phosphorylation of HDAC5. Increases in [Na(+)](i) activate SIK1 and MEF2 via a parallel increase in intracellular calcium through the reverse mode of Na(+)/Ca(2+)-exchanger and activation of CaMK1. These data obtained in a cardiac cell line suggest that increases in intracellular sodium could influence myocardial growth by controlling transcriptional activation and gene expression throughout the activation of the SIK1 network.