A novel poly (glycine) biosensor towards the detection of indigo carmine: A voltammetric study

The electrochemical behavior of indigo carmine (IC) at poly (glycine) modified carbon paste electrode (PGMCPE) was investigated by cyclic and differential pulse voltammetry. The oxidation peak of IC was observed in phosphate buffer of pH 6.5. The influence of different pH, scan rate, and concentration were analyzed. The probable reaction mechanism involved in the oxidation of IC was also proposed. Results showed that PGMCPE a remarkable electrocatalytic activity for the oxidation of IC under optimal conditions. The electrocatalytic response of the sensor was proportional to the IC concentration in the range of (2 × 10⁻⁶–1 × 10⁻⁵ M) and (1.5 × 10⁻⁵–6 × 10⁻⁵ M) with a limit of detection 11 × 10⁻⁸ M and limit of quantification 3.6 × 10⁻⁷ M. The modified electrode demonstrated many advantages such as simple preparation, high sensitivity, low detection of limit, excellent catalytic activity, short response time, and remarkable antifouling property toward IC and its oxidation product.     

Electrochemical determination of paraquat in citric fruit based on electrodeposition of silver particles onto carbon paste electrode

Carbon paste electrodes (CPEs) modified with silver particles present an interesting tool in the determination of paraquat (PQ) using square wave voltammetry. Metallic silver particle deposits have been obtained via electrochemical deposition in acidic media using cyclic voltammetry. Scanning electron microscopy and X-ray diffraction measurements show that the silver particles are deposited onto carbon surfaces in aggregate form. The response of PQ with modified electrode (Ag-CPE) related to Ag/CP loading, preconcentration time, and measuring solution pH was investigated. The result shows that the increase in the two cathodic peak currents (Peak 1 and Peak 2), under optimized conditions, was linear with the increase in PQ concentration in the range 1.0 × 10⁻⁷ mol/L to 1.0 × 10⁻³ mol/L. The detection limit and quantification limit were 2.01 × 10⁻⁸ mol/L and 6.073 × 10⁻⁸ mol/L, respectively for Peak 1. The precision expressed as relative standard deviation for the concentration level 1.0 × 10⁻⁵ mol/L (n = 8) was found to be 1.45%. The methodology was satisfactorily applied for the determination of PQ in citric fruit cultures.     

Cheap and easy modification of glassy carbon electrode for voltammetric determination of dopamine in the presence of ascorbic acid

Background and the purpose of the study

Different methods have been proposed to modify glassy carbon electrode in order to determine dopamine (DA), as one of the most important neurotransmitters in central nervous systems of mammalian. These methods are time comsuming and in some cases expensive. In this work, a very simple and cheap pretreatment method is developed for the bare glassy carbon electrode (GCE) to determine DA in the presence of Ascorbic acid (AA).

Methods

Cyclic voltammetry as an electrochemical activation procedure was used for activation of glassy carbon electrode in order to separate diffrential pulse peaks of DA and AA. The effect of different parameters such as pH for supporting electrolyte, range of potential and the number of cycles were investigated. Finally, differential pulse voltammetry was used to determine DA in the presence of AA.

Results

On the activated electrode under optimum condition, anodic peak of AA shifted to negative potentials and peak current decreased, but the peak current of DA increased. The peak current was linearly proportional to the bulk concentration of DA in the range of 6.5×10⁻⁷–1.8×10⁻⁵ mol l⁻¹. The limit of detection was 6.2×10⁻⁷ mol l⁻¹.

Conclusion

A simple and cheap method was developed for the activation of glassy carbon electrode. It was possible to determine DA in the presence of AA on the treated electrode. The proposed method was used to determine DA in pharmacutical samples.     

The H2 Receptor Antagonist Nizatidine is a P-Glycoprotein Substrate: Characterization of its Intestinal Epithelial Cell Efflux Transport

The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H₂ receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05–10 mM in both apical–basolateral (AP–BL) and BL–AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL–AP than AP–BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC₅₀ of verapamil on nizatidine P-gp secretion was 1.2 × 10⁻² mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J_max = 5.7 × 10⁻³ nmol∙cm⁻²∙s⁻¹ and K_m = 2.2 mM) and one nonsaturable component (K_d = 7 × 10⁻⁴ μL∙cm⁻²∙s⁻¹). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V_max and K_m estimated for nizatidine P-gp-mediated secretion were 4 × 10⁻³ nmol∙cm⁻²∙s⁻¹ and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug–drug interactions.Key words: BCS class III drugs, caco-2 permeability, efflux transporters, intestinal absorption, nizatidine, P-glycoprotein     

Testosterone ameliorates streptozotocin-induced memory impairment in male rats

Aim： To study the effects of testosterone on streptozotocin （STZ）-induced memory impairment in male rats.
Methods： Adult male Wistar rats were intracerebroventricularly （icv） infused with STZ （750 μg） on d 1 and d 3, and a passive avoidance task was assessed 2 weeks after the first injection of STZ. Castration surgery was performed in another group of rats, and the passive avoidance task was assessed 4 weeks after the operation. Testosterone （1 mg·kg^-1·d^-1, sc）, the androgen receptor antagonist flutamide （10 mg·kg^-1·d^-1, ip）, the estrogen receptor antagonist tamoxifen （1 mg·kg^-1·d^-1, ip） or the aromatase inhibitor letrozole （4 mg·kg^-1·d^-1, ip） were administered for 6 d after the first injection of STZ.
Results： STZ administration and castration markedly decreased both STL1 （the short memory） and STL2 （the long memory） in passive avoidance tests. Testosterone replacement almost restored the STL1 and STL2 in castrated rats, and significantly prolonged the STL1 and STL2 in STZ-treated rats. Administration of flutamide, letrozole or tamoxifen significantly impaired the memory in intact rats, and significantly attenuated the testosterone replacement in improving STZ- and castration-induced memory impairment.
Conclusion： Testosterone administration ameliorates STZ- and castration-induced memory impairment in male Wistar rats.     

The analytical determination and electrochemiluminescence behavior of amoxicillin

A novel electrochemiluminescence (ECL) luminophor of amoxicillin was studied and found to generate ECL following the oxidation or reduction of amoxicillin. The amoxicillin oxidation state was also found to eliminate the reduction state, generating ECL. When solutions of amoxicillin were scanned between +1.5 V and −1.0 V with a graphite electrode in the presence of cetyltrimethyl ammonium bromide using KC1 as the supporting electrolyte, ECL emissions were observed at potentials of −0.7 V and +0.5 V. The ECL intensity at −0.7 V was enhanced by H₂O₂. Based on these findings, an ECL method for the determination of the amoxicillin concentration is proposed. The ECL intensities were linear with amoxicillin concentrations in the range of 1.8 × 10⁻⁸ g/mL to 2.5 × 10⁻⁷ g/mL, and the limit of detection (signal/noise = 3) was 5 × 10⁻⁹ g/mL. The florescence of amoxicillin had the greatest emission intensity in a neutral medium, with the emission wavelength dependent on the excitation wavelength. The experiments on the ECL mechanism for amoxicillin found that the electrochemical oxidation products of dissolved oxygen and active oxygen species contributed to the ECL process. The data also suggest that the hydroxyl group of amoxicillin contributed to its ECL emission.     

Nonenzymatic glucose sensor based on disposable pencil graphite electrode modified by copper nanoparticles

A nonenzymatic glucose sensor based on a disposable pencil graphite electrode (PGE) modified by copper nanoparticles [Cu(NP)] was prepared for the first time. The prepared Cu(NP) exhibited an absorption peak centered at ~562 nm using UV-visible spectrophotometry and an almost homogenous spherical shape by scanning electron microscopy. Cyclic voltammetry of Cu(NP)-PGE showed an adsorption controlled charge transfer process up to 90.0 mVs⁻¹. The sensor was applied for the determination of glucose using an amperometry technique with a detection limit of [0.44 (±0.01) μM] and concentration sensitivity of [1467.5 (±1.3) μA/mMcm⁻²]. The preparation of the Cu(NP)-PGE sensor was reproducible (relative standard deviation = 2.10%, n = 10), very simple, fast, and inexpensive, and the Cu(NP)-PGE is suitable to be used as a disposable glucose sensor.     

Pharmacokinetics of panaxatrol disuccinate sodium, a novel anti-cancer drug from Panax notoginseng, in healthy volunteers and patients with advanced solid tumors

Aim:

To evaluate single-dose and multiple-dose pharmacokinetics of panaxatrol disuccinate sodium in healthy volunteers and patients with advanced solid tumors.

Methods:

In the single-dose pharmacokinetic study, 27 healthy volunteers received panaxatrol disuccinate sodium in three doses (70, 100, and 140 mg·m⁻²). In the multiple-dose pharmacokinetic study, Panaxatrol disuccinate sodium was administered to 8 patients at 100 mg·m⁻² daily in a 30-day continuous intravenous injection. Determination of the panaxatrol disuccinate sodium plasma concentration was performed by an LC-MS method. The pharmacokinetic analysis system — Drug and Statistics (DAS) — was applied to assess plasma panaxatrol disuccinate sodium concentration-time data.

Results:

After a single intravenous dose of 70, 100, or 140 mg·m⁻² was administered to subjects, panaxatrol disuccinate sodium distributed broadly, and the plasma concentration of panaxatrol disuccinate sodium declined rapidly. No significant differences were observed in the main pharmacokinetic parameters among the three dosing groups, including AUC_0–t, MRT_0–t, VRT_0–t, t_1/2Z, CL_z/F, V_z/F, and C₀ (P>0.05). In the multiple-dose pharmacokinetic study, the mean steady-state peak concentration (C_max), trough concentration (C_min), average concentration (C_av), mean steady state AUC (AUC_ss) and the degree of fluctuation were 13.96±15.48 mg·L⁻¹, 0.18±0.29 mg·L⁻¹, 0.15±0.29 mg·L⁻¹, 3.58±6.94 mg·L⁻¹·h, and 148.00±117.18, respectively. At any given dose of panaxatrol disuccinate sodium, interindividual variability in the pharmacokinetic parameters was obvious.

Conclusion:

The effect of the dose level on single-dose pharmacokinetics of panaxatrol disuccinate sodium was not significant. No accumulation was observed with exposure to 100 mg·m⁻² panaxatrol disuccinate sodium in the 30-day continuous intravenous injection. All subjects were evaluated for tolerability throughout the study. Thus, the phase II dose of panaxatrol disuccinate sodium may be considered to be 100 mg·m⁻² for a 30-day continuous intravenous injection to treat patients with advanced solid tumors.     

Single-dose pharmacokinetics of ampicillin and tobramycin administered by hypodermoclysis in young and older healthy volunteers

1To test the feasibility of administering antibiotics by subcutaneous infusion to the elderly, we compared the pharmacokinetics of tobramycin (single dose of 80 mg) given by hypodermoclysis (HDC) with the kinetics of the antibiotic injected intravenously (i.v.) in 10 young (<50 years old) and 10 elderly (>65 years old) healthy volunteers. Similar studies were performed with ampicillin (single dose of 1 g) in 12 young and 10 older healthy volunteers. 2Compared with the i.v. route, HDC delayed the time to reach the maximal plasma concentration (t_max) of tobramycin in young volunteers: 32±6 (s.d.) min vs 88±46, P<0.005, and older volunteers: 27±4 min vs 89±15, P<0.005. Administration of the antibiotics by HDC was well tolerated. The plasma concentration of tobramycin 30 min after the end of infusion (C₆₀) was lower (P<0.05) following HDC than after the i.v. route in both young, 2.2±0.7 vs 3.5±0.8 μg ml⁻¹, and elderly subjects, 2.2±0.8 vs 3.8±0.9. μg ml⁻¹. 3The area under the curve (AUC) of tobramycin given by HDC was slightly smaller than when given i.v., i.e. in young subjects: 740±225 (s.d.) vs 893±223 μg ml⁻¹ min, NS, and in the elderly: 980±228 vs 1056±315 μg ml⁻¹ min, NS. 4When ampicillin was administered by HDC, the t_max was also delayed in young volunteers: 45±18 vs 23±6 min, and in the elderly: 49±18 vs 27±4 min, P<0.005, the AUC was greater by HDC than i.v. in the young volunteers: 4527±1658 μg ml⁻¹ min vs 3810±1033 μg ml⁻¹ min and in the elderly: 6795±2094 μg ml⁻¹ min vs 4217±1518 μg ml⁻¹ min, and the C₆₀ was higher by HDC in the young: 27±7 vs 24±9 μg ml⁻¹, and in the elderly: 32±9 vs 23±11 μg ml⁻¹, P<0.05. 5In conclusion, HDC delays the entry of the antibiotic into the systemic circulation, but did not affect the amount available. HDC was well tolerated and could become an adequate method for antibiotic administration to the elderly.     

Pharmacokinetic and pharmacodynamic interaction trial after repeated oral doses of imdapril and digoxin in healthy volunteers

Aims To investigate the potential pharmacokinetic and pharmacodynamic interaction between imidapril and digoxin. Methods AUC, C_max and t_max of imidapril, imidaprilat and digoxin were calculated and evaluated in a randomized, doubleblind three-period cross-over design in 12 healthy volunteers after 8 days treatment with the following combinations: digoxin 0.25 mg day⁻¹+placebo (D+P); imidapril 10 mg day⁻¹+placebo (I+P); imidapril 10 mg day⁻¹+digoxin 0.25 mg day⁻¹ (I+D). Results Mean AUC (0, 24 h) of digoxin was 10.4 (±4.9 s.d.) ng ml⁻¹ h (D+P) and 10.7 (±3.9 s.d.) ng ml⁻¹ h (I+D), respectively (90%-confidence intervals [ CI] for the ratio of (D+P) and (I+D): 0.91–1.27, point estimator [PE]: 1.06). Mean AUC (0, 24 h) of imidapril was 133 (±86 s.d.) ng ml⁻¹ h (I+P) and 108 (±52 s.d.) ng ml⁻¹ h (I+D), respectively (90%-CI: 0.76–0.94, PE 0.85). AUC (0, 24 h) of imidaprilat was 215 (±91 s.d.) ng ml⁻¹ h (I+P) and 194 (±54 s.d.) ng ml⁻¹ h (I+D), respectively (90%-CI: 0.80–1.08, PE 0.93). C_max was 19.9 (±8.7 s.d.) ng ml⁻¹ (I+P) and 15.9 (±5.3 s.d.) ng ml⁻¹ (I+D) (90%-CI: 0.67–1.00, PE 0.82). The results indicate a slight reduction of imidapril and imidaprilat plasma levels when coadministered with digoxin without any effect on digoxin plasma levels. Maximal ACE-inhibition was 79% (I+P) and 67% (I+D). Conclusions Grouped data analysis of imidaprilat plasma levels vs ACE-activity showed that for maximal inhibition of plasma ACE activity, imidaprilat plasma levels should exceed 10 ng ml⁻¹. Under digoxin and imidapril, more plasma concentrations of imidaprilat were seen under this level as after imidapril alone, this reduces the integral of the ACE-inhibition/time curves by about 20 to 30%.     

Mechanisms mediating the ability of caffeine to influence MDMA (��Ecstasy��)-induced hyperthermia in rats

Background and purpose:

Caffeine exacerbates the hyperthermia associated with an acute exposure to 3,4 methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) in rats. The present study investigated the mechanisms mediating this interaction.

Experimental approach:

Adult male Sprague-Dawley rats were treated with caffeine (10 mg·kg⁻¹; i.p.) and MDMA (15 mg·kg⁻¹; i.p.) alone and in combination. Core body temperatures were monitored before and after drug administration.

Key results:

Central catecholamine depletion blocked MDMA-induced hyperthermia and its exacerbation by caffeine. Caffeine provoked a hyperthermic response when the catecholamine releaser d-amphetamine (1 mg·kg⁻¹) was combined with the 5-HT releaser d-fenfluramine (5 mg·kg⁻¹) or the non-selective dopamine receptor agonist apomorphine (1 mg·kg⁻¹) was combined with the 5-HT₂ receptor agonist DOI (2 mg·kg⁻¹) but not following either agents alone. Pretreatment with the dopamine D₁ receptor antagonist Schering (SCH) 23390 (1 mg·kg⁻¹), the 5-HT₂ receptor antagonist ketanserin (5 mg·kg⁻¹) or α₁-adreno- receptor antagonist prazosin (0.2 mg·kg⁻¹) blocked MDMA-induced hyperthermia and its exacerbation by caffeine. Co-administration of a combination of MDMA with the PDE-4 inhibitor rolipram (0.025 mg·kg⁻¹) and the adenosine A_1/2 receptor antagonist 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-C]quinazolin-5-amine 15943 (10 mg·kg⁻¹) or the A_2A receptor antagonist SCH 58261 (2 mg·kg⁻¹) but not the A₁ receptor antagonist DPCPX (10 mg·kg⁻¹) exacerbated MDMA-induced hyperthermia.

Conclusions and implications:

A mechanism comprising 5-HT and catecholamines is proposed to mediate MDMA-induced hyperthermia. A combination of adenosine A_2A receptor antagonism and PDE inhibition can account for the exacerbation of MDMA-induced hyperthermia by caffeine.     

Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones

Background and purpose:

The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn.

Experimental approach:

Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs).

Key results:

Icilin (3 or 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29°C), 3 µmol·L⁻¹ icilin (EC₅₀ 1.5 µmol·L⁻¹) and menthol (EC₅₀ 263 µmol·L⁻¹) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 µmol·L⁻¹ icilin (EC₅₀ 79 µmol·L⁻¹), allyl isothiocyanate (EC₅₀ 226 µmol·L⁻¹), cinnamaldehyde (EC₅₀ 38 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) increased miniature EPSC rate. The response to 100 µmol·L⁻¹, but not 3 µmol·L⁻¹ icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 µmol·L⁻¹, but not to 100 µmol·L⁻¹ icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 µmol·L⁻¹ icilin and innocuous cold were located more superficially than those responding to 100 µmol·L⁻¹ icilin.

Conclusions and implications:

Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.     

AVE8134, a novel potent PPARα agonist, improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats

Aim:

AVE8134 is a structurally novel potent PPARα agonist. The aim of this study is to investigate the efficacy of AVE8134 on lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.

Methods:

A cell based PPAR Gal4 transactivation assay was constructed for testing the activities of AVE8134 at 3 different PPAR isoforms in vitro. Transgenic human Apo A1 (hApo A1) mice and insulin-resistant ZDF rats were used to evaluate the effects of AVE8134 in vivo.

Results:

AVE8134 was a full PPARα dominated PPAR agonist (the values of EC₅₀ for human and rodent PPARα receptor were 0.01 and 0.3 μmol/L, respectively). AVE8134 was not active at PPARδ receptor. In female hApo A1 mice, AVE8134 (1–30 mg·kg⁻¹·d⁻¹, po for 12 d) dose-dependently lowered the plasma triglycerides, and increased the serum HDL-cholesterol, hApo A1 and mouse Apo E levels. In female ZDF rats, AVE8134 (3–30 mg·kg⁻¹·d⁻¹ for 2 weeks) improved insulin-sensitivity index. In pre-diabetic male ZDF rats (at the age of 7 weeks), AVE8134 (10 mg·kg⁻¹·d⁻¹ for 8 weeks) produced an anti-diabetic action comparable to rosiglitazone, without the PPARγ mediated adverse effects on body weight and heart weight. In male ZDF rats (at the age of 6 weeks), AVE8134 (20 mg·kg⁻¹·d⁻¹ for 12 weeks) increased mRNA levels of the target genes LPL and PDK4 about 20 fold in the liver, and there was no relevant effect with rosiglitazone.

Conclusion:

AVE8134 improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.     

Morphine, morphine-6-glucuronide and morphine-3-glucuronide pharmacokinetics in newborn infants receiving diamorphine infusions

1The pharmacokinetics of morphine, morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) were studied in 19 ventilated newborn infants(24–41 weeks gestation) who were given a loading dose of 50 μg kg⁻¹ or 200 μg kg⁻¹ of diamorphine followed by an intravenous infusion of 15 μg kg⁻¹ h⁻¹ of diamorphine. Plasma concentrations of morphine, M3G and M6G were measured during the accrual to steady-state and at steady state of the diamorphine infusion. 2Following both the 50 μg kg⁻¹ or 200 μg kg⁻¹ loading doses the mean steady-state plasma concentration (±s.d.) of morphine, M3G and M6G were 86±52 ng ml⁻¹, 703±400 ng ml⁻¹ and 48±28 ng ml⁻¹ respectively and morphine clearance was found to be 4.6±3.2 ml min⁻¹ kg⁻¹. 3M3G formation clearance was estimated to be 2.5±1.8 ml min⁻¹ kg⁻¹, and the formation clearance of M6G was estimated to be 0.46±0.32 ml min⁻¹ kg⁻¹. 4M3G metabolite clearance was 0.46±0.60 ml min⁻¹ kg⁻¹, the elimination half-life was 11.1±11.3 h and the volume of distribution was 0.55±1.13 l kg⁻¹. M6G metabolite clearance was 0.71±0.36 ml min⁻¹ kg⁻¹, the elimination half-life was 18.2±13.6 h and the volume of distribution was 1.03±0.88 l kg⁻¹. 5No significant effect of the loading dose (50 μg kg⁻¹ or 200 μg kg⁻¹) on the plasma morphine or metabolite concentrations or their derived pharmacokinetic parameters was found. 6We were unable to identify correlations between gestational age of the infants and any of the determined pharmacokinetic parameters. 7M3G:morphine and M6G:morphine steady-state plasma concentration ratios were 11.0±10.8 and 0.8±0.8, respectively. 8The metabolism of morphine in neonates, in terms of the respective contributions of each glucuronide pathway, was similar to that in adults.     

Inositol trisphosphate-dependent Ca2+ stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland

Background and purpose

Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca²⁺ compartments in regulating slow wave activity in the guinea pig prostate.

Experimental approach

Standard intracellular membrane potential recording techniques were used.

Key results

The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L⁻¹) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L⁻¹) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP₃R)-mediated Ca²⁺ release with 2-aminoethoxy-diphenylborate (60 µmol·L⁻¹) or Xestospongin C (3 µmol·L⁻¹) or inhibiting phospholipase C and IP₃ formation using {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (5 µmol·L⁻¹) or neomycin (1 and 4 mmol·L⁻¹) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L⁻¹), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L⁻¹) or rotenone (10 µmol·L⁻¹), depolarized the membrane (8–10 mV) before abolishing electrical activity.

Conclusion and implications

These results suggest that slow wave activity was dependent on the cyclical release of Ca²⁺ from IP₃-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland.     

Nanomechanical analysis of insulinoma cells after glucose and capsaicin stimulation using atomic force microscopy

Aim:

Glucose stimulates insulin secretion from pancreatic islet β cells by altering ion channel activity and membrane potential in the β cells. TRPV1 channel is expressed in the β cells and capsaicin induces insulin secretion similarly to glucose. This study aims to investigate the biophysical properties of the β cells upon stimulation of membrane channels using an atomic force microscopic (AFM) nanoindentation system.

Methods:

ATCC insulinoma cell line was used. Cell stiffness, a marker of reorganization of cell membrane and cytoskeleton due to ion channel activation, was measured in real time using an integrated AFM nanoindentation system. Cell height that represented structural changes was simultaneously recorded along with cell stiffness.

Results:

After administration of glucose (16, 20 and 40 mmol/L), the cell stiffness was markedly increased in a dose-dependent manner, whereas cell height was changed in an opposite way. Lower concentrations of capsaicin (1.67×10⁻⁹ and 1.67×10⁻⁸ mol/L) increased the cell stiffness without altering cell height. In contrast, higher concentrations of capsaicin (1.67×10⁻⁶ and 1.67×10⁻⁷ mol/L) had no effect on the cell physical properties.

Conclusion:

A unique bio-nanomechanical signature was identified for characterizing biophysical properties of insulinoma cells upon general or specific activation of membrane channels. This study may deepen our understanding of stimulus-secretion coupling of pancreatic islet cells that leads to insulin secretion.     

Preclinical characterization of WAY-211612: a dual 5-HT uptake inhibitor and 5-HT1A receptor antagonist and potential novel antidepressant

Background and purpose

As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT_1A receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT_1A receptor antagonist activities, was evaluated in preclinical models.

Experimental approach

Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models.

Key results

WAY-211612 inhibited 5-HT reuptake (K_i = 1.5 nmol·L⁻¹; K_B = 17.7 nmol·L⁻¹) and exhibited full 5-HT_1A receptor antagonist activity (K_i = 1.2 nmol·L⁻¹; K_B = 6.3 nmol·L⁻¹; I_max 100% in adenyl cyclase assays; K_B = 19.8 nmol·L⁻¹; I_max 100% in GTPγS). WAY-211612 (3 and 30 mg·kg⁻¹, po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT_1A receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3–30 mg·kg⁻¹, po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg·kg⁻¹, s.c.) and a 5-HT_1A antagonist (WAY-100635; 0.3 mg·kg⁻¹, s.c). WAY-211612 (3.3–30 mg·kg⁻¹, s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3–30 mg·kg⁻¹, i.p. and 10–56 mg·kg⁻¹, po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg·kg⁻¹, i.p.) in the rat scheduled-induced polydipsia model.

Conclusions and implications

These findings suggest that WAY-211612 may represent a novel antidepressant.     

Evodiamine improves congnitive abilities in SAMP8 and APP(swe)/PS1(ΔE9) transgenic mouse models of Alzheimer's disease

Aim:

To investigate the effect of evodiamine (a quinolone alkaloid from the fruit of Evodia rutaecarpa) on the progression of Alzheimer''s disease in SAMP8 and APP^swe/PS1^ΔE9 transgenic mouse models.

Methods:

The mice at age of 5 months were randomized into the model group, two evodiamine (50 mg·kg⁻¹·d⁻¹ and 100 mg·kg⁻¹·d⁻¹) groups and an Aricept (2 mg·kg⁻¹·d⁻¹) group. The littermates of no-transgenic mice and senescence accelerated mouse/resistance 1 mice (SAMR1) were used as controls. After 4 weeks of treatment, learning abilities and memory were assessed using Morris water-maze test, and glucose uptake by the brain was detected using positron emission tomography/computed tomography (PET/CT). Expression levels of IL-1β, IL-6, and TNF-α in brain tissues were detected using ELISA. Expression of COX-2 protein was determined using Western blot.

Results:

In Morris water-maze test, evodiamine (100 mg·kg⁻¹·d⁻¹) significantly alleviated the impairments of learning ability and memory. Evodiamine (100 mg·kg⁻¹·d⁻¹) also reversed the inhibition of glucose uptake due to development of Alzheimer''s disease traits in mice. Furthermore, the dose of evodiamine significantly decreased the expression of IL-1β, IL-6, TNF-α, and COX-2 that were involved in the inflammation due to Alzheimer''s disease.

Conclusion:

The results indicate that evodiamine (100 mg·kg⁻¹·d⁻¹) improves cognitive abilities in the transgenic models of Alzheimer''s disease.     

Disposition of diazepam in young and elderly subjects after acute and chronic dosing

1. The pharmacokinetics of diazepam were examined in seven young (20–30 years) and six elderly (60–75 years) males prior to and also after chronic oral dosing of diazepam.
2. Following intravenous administration, the half-life and volume of distribution of ¹⁴C-labelled diazepam in the elderly were approximately twofold greater than corresponding estimates in younger subjects (mean ±s.d., 71.5±27.6 vs 44.5±16.5 h and 1.39±0.32 vs 0.88±0.30 1 kg⁻¹, respectively). Clearance did not differ between the two groups (0.26±0.09 vs 0.29±0.09 ml min⁻¹ kg⁻¹).
3. The accumulation of diazepam and its major metabolite, desmethyldiazepam, were extensive during chronic administration. A radioreceptor assay that measured total benzodiazepine activity, including diazepam and its active metabolites, indicated that the accumulation of ‘benzodiazepine equivalents’ was similar to the sum of the accumulated diazepam and desmethyldiazepam concentration levels. However, the level of ‘benzodiazepine equivalents’ on multiple-dosing was about double that of the predicted steady-state ‘equivalent’ concentration from single-dose studies. This was due to the insensitivity of the radioreceptor assay for desmethyldiazepam following single-dose diazepam administration.
4. There were no age- or dosing-related differences in diazepam clearance (0.37±0.22 vs 0.32±0.18 ml min⁻¹ kg⁻¹, young vs elderly, single-dose; 0.37± 0.11 vs 0.27±0.12 ml min⁻¹ kg⁻¹, young vs elderly, multiple-dose) and no age-related differences in the levels of accumulated ‘benzodiazepine equivalents’ (243.7±60.1 vs 288.0±125.8 ng ml⁻¹, young vs elderly).

     

Chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice

BACKGROUND AND PURPOSE

ApolipoproteinE-deficient [apoE (−/−)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (−/−) mice.

EXPERIMENTAL APPROACH

ApoE (−/−) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg⁻¹·h⁻¹) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg⁻¹·h⁻¹) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys.

KEY RESULTS

Ang-(1-7)-treated apoE (−/−) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (−/−) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (−/−) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased.

CONCLUSION AND IMPLICATIONS

Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.