Human skin penetration of cobalt nanoparticles through intact and damaged skin

Cobalt nanoparticles (CoNPs) are produced for several industrial and biomedical applications but there is a lack of data on human cutaneous absorption. Cobalt is also a skin sensitizer that can cause allergic contact dermatitis. Co applied as NPs, due to their small size and high surface, can penetrate into the skin in higher amount that bulk material. The aim of this study was to evaluate the absorption of Co applied as NPs in both intact and damaged skin. Experiments were performed using Franz cells and 1.0 mg cm^?2 of CoNPs was applied as donor phase for 24 h. Mean Co content of 8.5 ± 1.2 ng cm^?2 and 1.87 ± 0.86 μg cm^?2 were found in the receiving solutions of Franz cells when the CoNPs suspension was applied on intact skin and on damaged skin, respectively. Twenty-four hours Co flux permeation was 76 ± 49 ng cm^?2 h^?1 in damaged skin with a lag time of 2.8 ± 2.1 h. This study suggests that Co applied as NPs is able to penetrate the human skin in an in vitro diffusion cell system.     

Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells

IntroductionThe major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs.MethodsThe purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical–basolateral (A–B) and basolateral–apical (B–A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively.ResultsThe apparent permeability (P_app) results for the A–B direction were: P_{app FLU A–B} = 0.54 × 10^? 4 cm·s^? 1, P_{app METO A–B} = 7.99 × 10^? 4 cm·s^? 1, P_{app 3TC A–B} = 4.58 × 10^? 4 cm·s^? 1 and P_{app AZT A–B} = 5.34 × 10^? 4 cm·s^? 1. For the B–A direction, the P_app results were: P_{app FLU B–A} = 0.56 × 10^? 4 cm·s^? 1, P_{app METO B–A} = 0.25 × 10^? 4 cm·s^? 1, P_{app 3TC B–A} = 0.24 × 10^? 4 cm·s^? 1 and P_{app AZT B–A} = 0.19 × 10^? 4 cm·s^? 1.DiscussionFor the A–B direction, the P_app results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A–B direction, the P_app results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B–A direction the P_app results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification.     

The role of Na+, K+-ATPase in the hypoxic vasoconstriction in isolated rat basilar artery

Hypoxia-induced cerebrovascular dysfunction is a key factor in the occurrence and the development of cerebral ischemia. Na⁺, K⁺-ATPase affects the regulation of intracellular Ca^2 + concentration and plays an important role in vascular smooth muscle function. However, the potential role of Na⁺, K⁺-ATPase in hypoxia-induced cerebrovascular dysfunction is unknown. In this study, we found that the KCl-induced contraction under hypoxia in rat endothelium-intact basilar arteries is similar to that of denuded arteries, suggesting that hypoxia may cause smooth muscle cell (SMC)-dependent vasoconstriction in the basilar artery. The Na⁺, K⁺–ATPase activity of the isolated basilar artery with or without endothelium significantly reduced with prolonged hypoxia. Blocking the Na⁺–Ca^2 + exchanger with Ni^2 + (10^− 3 M) or the L-type Ca^2 + channel with nimodipine (10^− 8 M) dramatically attenuated KCl-induced contraction under hypoxia. Furthermore, prolonged hypoxia significantly reduced Na⁺, K⁺-ATPase activity and increased [Ca^2 +]_i in cultured rat basilar artery SMCs. Hypoxia reduced the protein and mRNA expression of the α₂ isoform of Na⁺, K⁺-ATPase in SMCs in vitro. We used a low concentration of the Na⁺, K⁺-ATPase inhibitor ouabain, which possesses a high affinity for the α₂ isoform. The contractile response in the rat basilar artery under hypoxia was partly inhibited by ouabain pretreatment. The decreased Na⁺, K⁺-ATPase activity in isolated basilar artery and the increased [Ca^2 +]_i in SMCs induced by hypoxia were partly inhibited by pretreatment with a low concentration of ouabain. These results suggest that hypoxia may educe Na⁺, K⁺-ATPase activity in SMCs through the α₂ isoform contributing to vasoconstriction in the rat basilar artery.     

The effects of sulfur mustard exposure and freezing on transdermal penetration of tritiated water through ex vivo pig skin

The percutaneous absorption of tritiated water (³H₂O) through sulfur mustard (SM) exposed abdominal pig skin was measured using in vitro Franz-type static diffusion cells. The barrier function to water permeation following exposure to liquid SM for 8 min and excision 3 h later did not change significantly. A small, but statistically significant difference (P < 0.05) in steady state penetration (Jss), permeability coefficient (Kp) and lag time (t_L) of ³H₂O was observed between fresh skin and skin stored frozen (?20 °C) for up to two weeks. Steady-state penetration and Kp values were significantly higher (P < 0.05) in skin stored frozen compared with fresh skin. Fresh naïve skin had an average Kp of 1.65 × 10^?3 cm h^?1, whereas frozen naïve skin was 2.04 × 10^?3 cm h^?1. Fresh SM exposed skin had a mean Kp of 1.72 × 10^?3 cm h^?1, whereas frozen SM exposed skin was 2.31 × 10^?3 cm h^?1. Lag times were also shorter (P < 0.05) in skin that had been stored frozen. Frozen, SM-exposed porcine abdominal skin may be used for in vitro penetration studies, but effects of treatment and storage on the barrier layer should be taken into account.     

Air pollution-related metals induce differential cytokine responses in bronchial epithelial cells

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd^2 +, Zn^2 + and As^3 + induced CXCL8 and IL-6 release at concentrations below 100 μM, and Mn^2 + and Ni^2 + at concentrations above 200 μM. In contrast, VO₄^3 −, Cu^2 + and Fe^2 + did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd^2 +, Zn^2 + and As^3 + induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn^2 +- and As^3 +-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd^2 +, Zn^2 + and As^3 + seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.     

Screening quality for Ca2 +-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin

IntroductionIonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca^2 + concentration ([Ca^2 +]_i). This difficulty prevents obtaining high-quality data regarding Ca^2 +-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca^2 +]_i in the IonWorks Quattro automated patch clamp system to evaluate BK channels.MethodsElectrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca^2 +]_i, ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca^2 + chelator, BAPTA-AM (20 μM).ResultsBK current activated by step pulses to + 100 mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11 nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1 Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (> 20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10 μM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30 μM.DiscussionThe main finding of this study is that the [Ca^2 +]_i variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca^2 +-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca^2 +-activated ion channels such as SK channels.     

Effect of the ostreolysin A/pleurotolysin B pore-forming complex on intracellular Ca2 + activity in the vascular smooth muscle cell line A10

Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca^2 + indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca^2 + activity [Ca^2 +]_i in A10 cells. This effect was absent in a Ca^2 +-free medium, indicating that OlyA/PlyB-induced [Ca^2 +]_i increase was dependent on Ca^2 + influx into cells. The increase in [Ca^2 +]_i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La^3 +, ii) the inhibitor of the sodium–calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca^2 +-ATPase inhibitor thapsigargin reduced the [Ca^2 +]_i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K⁺ in the medium did not prevent OlyA/PlyB-induced [Ca^2 +]_i. In summary, our data could suggest that the OlyA/PlyB-induced increase in [Ca^2 +]_i is due to an influx of Ca^2 + through a variety of co-existing plasma membrane Ca^2 +-permeable channels, Ca^2 + entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca^2 + release, altogether leading to disturbed Ca^2 + homeostasis in A10 cells.     

Covalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k_inact/K_i) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10³ M^?1 s^?1 and 4.5 (±1.3) × 10³ M^?1 s^?1, respectively]. However, k_inact/K_i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10² M^?1 s^?1 and 3.9 (±1.0) × 10³ M^?1 s^?1, respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.     

The pharmacokinetics of orally administered S-carboxymethyl-l-cysteine in the dog,calf and sheep

The aim of this study was to investigate the feasibility of employing S-carboxymethyl-l-cysteine as a treatment of chronic obstructive pulmonary disease in dogs. To this end the pharmacokinetic parameters of orally administered S-carboxymethyl-l-cysteine were determined in the dog, cow and sheep. Six healthy beagle dogs, six endogenous Greek sheep and four Holstein Fresian calves were orally dosed with 10 mg/kg body weight of S-carboxymethyl-l-cysteine. No significant differences in T_max and T_1/2 were reported between the species. However, significantly higher AUC_(0–last), 21.56 ± 6.67 μg h ml^?1 and AUC_(0–∞), 21.63 ± 6.68 μg h ml^?1 were seen in the dogs compared to the sheep and calves. The calculated V_D was significantly higher in the sheep (10.4 ± 2.7 L kg^?1) and the calves (3.8 ± 0.7 L kg^?1) compared to the dogs (1.0 ± 0.6 L kg^?1). The rank order of increasing C_L was sheep (3.4 ± 2.7 L h^?1 kg^?1) > calves (2.7 ± 0.4 L h^?1 kg^?1) > dogs (0.5 ± 0.2 L h^?1 kg^?1). The result for the dogs was significantly lower that the calculated C_L for the sheep and calves.All these results indicate that the oral administration of S-carboxymethyl-l-cysteine may be useful during the therapeutic management of chronic obstructive pulmonary disease in dogs.     

Using transdermal iontophoresis to increase granisetron delivery across skin in vitro and in vivo: Effect of experimental conditions and a comparison with other enhancement strategies

The objectives of the study were (i) to investigate the effect of experimental parameters on the iontophoretic transport of granisetron, (ii) to identify the relative contributions of electromigration (EM) and electroosmosis (EO), (iii) to determine the feasibility of delivering therapeutic amounts of drug for the treatment of chemotherapy-induced nausea and vomiting and (iv) to test the in vitro results in a simple animal model in vivo. Preliminary in vitro studies using aqueous granisetron formulations investigating the effect of drug concentration (5, 10, 20 and 40 mM) and current density (0.1, 0.2, 0.3 mA cm^?2) were performed using porcine ear skin. As expected, cumulative delivery in vitro at the 20 and 40 mM concentrations was significantly greater than that at 5 and 10 mM, which were not statistically different (p < 0.05). Increasing the applied current density from 0.1 to 0.3 mA cm^?2 resulted in a ～4.2-fold increase in iontophoretic flux. Furthermore, in the absence of Na⁺ in the formulation, no dependence of iontophoretic flux on drug concentration was reported (at a granisetron concentration of 40 mM, the transport rate was 2.93 ± 0.62 μg cm^?2 min^?1). Co-iontophoresis of acetaminophen was used to show that EM was the predominant transport mechanism accounting for 71–86% of total granisetron delivery. In vivo studies in Wistar rats (40 mM granisetron; application of 0.3 mA cm^?2 for 5 h with Ag/AgCl electrodes and salt bridges) showed an average iontophoretic input rate (k_input) of 0.83 ± 0.26 μg min^?1 and a maximum plasma concentration (C_max) of 0.092 ± 0.004 μg ml^?1. Based on these results and given the known pharmacokinetics, transdermal iontophoresis could achieve therapeutic drug levels for the management of chemotherapy-induced emesis using a reasonably sized (4–6 cm²) patch.     

Arachidonic acid-evoked Ca2 + signals promote nitric oxide release and proliferation in human endothelial colony forming cells

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca^2 + concentration ([Ca^2 +]_i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca^2 + signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca^2 +]_i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca^2 +]_i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca^2 + signals required both intracellular Ca^2 + release and external Ca^2 + inflow. AA-induced Ca^2 + release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca^2 + entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca^2 + entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca^2 +]_i and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca^2 +- and NO-dependent manner. Therefore, AA-evoked Ca^2 + signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.     

Participation of the TRP channel in the cardiovascular effects induced by carvacrol in normotensive rat

Carvacrol has been described as an agonist/antagonist of different transient receptor potential (TRP) channels and voltage-dependent calcium channels (Ca_vs). The aim of this study was to evaluate the role of Ca_v and TRP channels following carvacrol stimulation. Initially, in mesenteric artery rings carvacrol relaxed phenylephrine-induced contractions. Furthermore, carvacrol inhibited contraction elicited by CaCl₂ in depolarizing nominally without Ca^2 + medium and antagonized the contractions induced by S(−)-Bay K 8644 and inhibited Ca^2 + currents indicating the inhibition of Ca^2 + influx through L-type Ca_v. Additionally, carvacrol antagonized the contractions induced by CaCl₂ in the presence of nifedipine/Cyclopiazonic acid/phenylephrine or nifedipine/Cyclopiazonic acid/KCl 60, suggesting a possible inhibition of calcium influx by store operated channels (SOCs), receptor operated channels (ROCs) and/or TRP channels. Interestingly, among the TRP channel blockers used, the effect induced by carvacrol was attenuated by Mg^2 + and potentiated by La^3 + and Gd^3 +, suggesting that TRP channels are involved in relaxation induced by carvacrol. Monoterpene also induced hypotension and bradycardia in non-anesthetized normotensive rats and negative inotropic and chronotropic effects. In conclusion, these results suggest that the hypotensive effect of carvacrol is probably due to bradycardia and a peripheral vasodilatation that involves, at least, the inhibition of the Ca^2 + influx through Ca_v and TRP channels.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

High Shear Rheology and Anisotropy in Concentrated Solutions of Monoclonal Antibodies

The high shear rheology of three concentrated solutions of immunoglobulin G1 monoclonal antibodies (mAbl, mAb2, and mAb3), differing only in their complementarity determining regions, was characterized using rotary and capillary rheometry. The more viscous solutions (mAb1 and mAb3) showed non-Newtonian behavior at high shear rates exhibiting both shear thinning and appreciable normal stress differences (NSDs) in the shear rate range γ = 10 to 10⁴ s^? 1. The rheograms were retraced after γ is increased and decreased, suggesting reversible self-associations under shear. In contrast, mAb2 solutions showed Newtonian behavior up to γ = 6 × 10⁴ s^? 1. The critical shear stress τ_c, corresponding to the onset of the reduction in the viscosity η, is a measure of mAb equilibrium cluster strength and increased rapidly with concentration for the high viscosity mAb solutions above 100 mg/mL. In addition, decreasing the temperature from 20 °C to 5 °C increased η at low γ, but shear-thinning was enhanced and its onset occurred at a lower γ_c. Using an Arrhenius model η = A exp(E_a/kT), the activation energy for viscous flow E_a was found to decrease for mAb1 solutions as γ was increased from 10 to 10⁴ s^? 1, suggesting mAb cluster disruption or rearrangement under shear. In contrast, for mAb2, this E_a remained constant in the γ range. Finally, mAb1 and mAb3 solutions showed appreciable NSDs, with their N₁ > 0 scaling linearly with γ in the range 10³ to 10⁴ s^? 1, whereas their |N₂/N₁| was less than 0.25 in this region. These suggest anisotropy and deformation of their solution microstructure toward the extensional quadrant of the flow at high γ. In contrast, the NSDs for mAb2 were close to zero indicating that the solution microstructure under shear is practically isotropic. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2538–2549, 2013     

The anti-diabetic drug repaglinide induces vasorelaxation via activation of PKA and PKG in aortic smooth muscle

We investigated the vasorelaxant effect of repaglinide and its related signaling pathways using phenylephrine (Phe)-induced pre-contracted aortic rings. Repaglinide induced vasorelaxation in a concentration-dependent manner. The repaglinide-induced vasorelaxation was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor (L-NAME) and a small-conductance Ca^2 +-activated K⁺ (SK_Ca) channel inhibitor (apamin) did not alter the vasorelaxant effect of repaglinide on endothelium-intact arteries. Pretreatment with an adenylyl cyclase inhibitor (SQ 22536) or a PKA inhibitor (KT 5720) effectively reduced repaglinide-induced vasorelaxation. Also, pretreatment with a guanylyl cyclase inhibitor (ODQ) or a PKG inhibitor (KT 5823) inhibited repaglinide-induced vasorelaxation. However, pretreatment with a voltage-dependent K⁺ (Kv) channel inhibitor (4-AP), ATP-sensitive K⁺ (K_ATP) channel inhibitor (glibenclamide), large-conductance Ca^2 +-activated K⁺ (BK_Ca) channel inhibitor (paxilline), or the inwardly rectifying K⁺ (Kir) channel inhibitor (Ba^2 +) did not affect the vasorelaxant effect of repaglinide. Furthermore, pretreatment with a Ca^2 + inhibitor (nifedipine) and a sarco-endoplasmic reticulum Ca^2 +-ATPase (SERCA) inhibitor (thapsigargin) did not affect the vasorelaxant effect of repaglinide. The vasorelaxant effect of repaglinide was not affected by elevated glucose (50 mM). Based on these results, we conclude that repaglinide induces vasorelaxation via activation of adenylyl cyclase/PKA and guanylyl cyclase/PKG signaling pathways independently of the endothelium, K⁺ channels, Ca^2 + channels, and intracellular Ca^2 + ([Ca^2 +]_i).     

Effect of cadmium on the extracellular Na+, K+, and Ca2+ in the gill and small intestine of Goldfish Carassius auratus

In this study, the toxic effect of cadmium on extracellular Na⁺, K⁺, and Ca²⁺ in the gill and small intestine of goldfish Carassius auratus was determined with the technique of ion chromatograph. Two-way ANOVA indicated that the two factors (Cd²⁺ treatment and time) and the interaction factor had significant effect on the level of Na⁺, K⁺, and Ca²⁺ in the small intestine and gill. 1.0 mg/L Cd²⁺ significantly increased Ca²⁺ level in the small intestine, but Ca²⁺ level in the gill was significantly decreased by 1.0 and 5.0 mg/L Cd²⁺ at 24, 48, and 72 h. Na⁺ and K⁺ level in the small intestine and gill was increased by 1.0 mg/L Cd²⁺ at three time points, but increased by 5.0 mg/L Cd²⁺ at a certain different time. In addition, Na⁺ level was significantly decreased by 5.0 mg/L Cd²⁺ at 24 or 48 h in the small intestine and gill. The results indicated that Cd²⁺ played an important role in regulating the level of Na⁺, K⁺, and Ca²⁺ in the small intestine and gill of goldfish C. auratus. A method was constructed to investigate the extracellular Na⁺, K⁺ and Ca²⁺ in the tissues of gold fish with ion chromatography.     

Stimulation of calcium-sensing receptors induces endothelium-dependent vasorelaxations via nitric oxide production and activation of IKCa channels

Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K⁺ channels in these responses. In wire myography studies, increasing [Ca^2 +]_o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca^2 +]_o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca^2 +]_o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca^2 +-activated K⁺ channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca^2 +]_o-induced vasorelaxations. Increasing [Ca^2 +]_o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations.     

A higher frequency of CD4+ CXCR5+ T follicular helper cells in patients with newly diagnosed Henoch–Schönlein purpura nephritis

T follicular helper (TFH) cells play an important role in the humoral immune responses. The aim of this study was to examine the frequency of different subsets of CD4⁺ CXCR5⁺ TFH cells and B cells in patients with new-onset Henoch–Schönlein purpura nephritis (HSPN). The numbers of different subsets of CD4⁺ CXCR5⁺ TFH cells, B cells and the constituents of serum cytokines were detected in a total of 25 patients with newly diagnosed HSPN before and after treatment, and in 14 healthy controls (HC). The potential connection of these cells with the clinical characteristics in HSPN patients was analyzed. The numbers of circulating CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ ICOS⁺ and CD4⁺ CXCR5⁺ PD-1⁺ TFH cells, CD86⁺ CD19⁺, CD38⁺ CD19⁺ B cells and serum IL-2, IL-4, IL-17A, IL-21 and IFN-γ were significantly higher in HSPN patients (p < 0.05) than in HC. Before and after treatment the numbers of CD4⁺ CXCR5⁺ TFH cells were negatively correlated with the values of eGFR (r = − 0.7162, p < 0.05; r = − 0.732, p < 0.05, respectively). Similarly the numbers of CD4⁺ CXCR5⁺ PD-1⁺ TFH cells were negatively correlated with 24-h urinary proteins (r = − 0.4013, p < 0.05; r = − 0.7857, p < 0.05, respectively), and the numbers of CD4⁺ CXCR5⁺ ICOS⁺ TFH cells were positively correlated with the levels of serum IL-21 (r = 0.5186, p < 0.05; r = 0.8503, p < 0.05, respectively) and 24-h urinary protein (r = 0.6045, p < 0.05; r = 0.833, p < 0.05, respectively) in these patients, regardless of treatment. Following treatment the numbers of CD4⁺ CXCR5⁺, CD4⁺ CXCR5⁺ PD-1⁺, and CD4⁺ CXCR5⁺ ICOS⁺ TFH cells, as well as serum levels of IL-21 were significantly reduced, however IL-4 levels were noticeably increased (p < 0.05). A higher frequency of circulating CD4⁺ CXCR5⁺ TFH cells existed in patients with HSPN and may be a viable therapeutic target.     

Mercury modulates the cytochrome P450 1a1, 1a2 and 1b1 in C57BL/6J mice: in vivo and in vitro studies

In the current study C57BL/6J mice were injected intraperitoneally with Hg^2 + in the absence and presence of TCDD. After 6 and 24 h the liver was harvested and the expression of Cyps was determined. In vitro, isolated hepatocytes were incubated with TCDD in the presence and absence of Hg^2 +. At the in vivo level, Hg^2 + significantly decreased the TCDD-mediated induction of Cyps at 6 h while potentiating their levels at 24 h. In vitro, Hg^2 + significantly inhibited the TCDD-mediated induction of Cyp1a1 in a concentration- and time-dependent manner. Interestingly, Hg^2 + increased the serum hemoglobin (Hb) levels in mice treated for 24 h. Upon treatment of isolated hepatocytes with Hb alone, there was an increase in the AhR-dependent luciferase activity with a subsequent increase in Cyp1a1 protein and catalytic activity levels. Importantly, when hepatocytes were treated for 2 h with Hg^2 + in the presence of TCDD, then the medium was replaced with new medium containing Hb, there was potentiation of the TCDD-mediated effect. In addition, Hg^2 + increased heme oxygenase-1 (HO-1) mRNA, which coincided with a decrease in the Cyp1a1 activity level. When the competitive HO-1 inhibitor, tin mesoporphyrin was applied to the hepatocytes there was a partial restoration of Hg^2 +-mediated inhibition of Cyp1a1 activity. In conclusion, we demonstrate for the first time that there is a differential modulation of the TCDD-mediated induction of Cyp1a1 by Hg^2 + in C57BL/6J mice livers and isolated hepatocytes. Moreover, this study implicates Hb as an in vivo specific modulator of Cyp1 family.     

In vivo sustained dermal delivery and pharmacokinetics of interferon alpha in biphasic vesicles after topical application

Biphasic vesicles, a novel nanostructured lipid-based delivery system show potential for topical application of interferon alpha (IFN α) for the treatment of human papillomavirus (HPV) infections (anogenital warts). Dermal delivery of IFN α encapsulated in biphasic vesicles (BPV-IFN α), applied topically to the skin, was characterized in a guinea pig model.BPV-IFN α (1 g, 2 MIU/g) was topically applied either as a single or multiple treatments on the skin of guinea pigs. As a comparison with currently used regimens, IFN α solution was administered intravenously or intradermally. Skin and serum samples were collected over 96 h, IFN α levels were determined by an antiviral assay, and half-life (t_1/2) and elimination (k) rates were calculated.Topical BPV-IFN α treatment resulted in maximum skin levels (about 100,000 U/100 cm²) of IFN α within 6 h and maintained for 72–96 h. Clearance from the skin after intradermal injections was initially fast (t_1/2 0.62 h, k 1.1179 h⁻¹), followed by a slower steady decrease after 6 h. After intravenous and intradermal administration, IFN α was rapidly cleared from the serum, t_1/2 0.75 h, k 0.9271 h⁻¹ and t_1/2 1.28 h, k 0.5421 h⁻¹, respectively, whereas after topical application, IFN α levels remained below 100 U/mL. Topical application of BPV- IFN α resulted in sustained delivery of biologically active IFN α locally into skin with minimal systemic exposure.