Determination of acidic non-steroidal anti-inflammatory drugs in aquatic samples by liquid chromatography-triple quadrupole mass spectrometry combined with carbon nanotubes-based solid-phase extraction.

A solid-phase extraction (SPE) method based on multi-walled carbon nanotubes (CNT) was developed for the determination of 12 acidic non-steroidal anti-inflammatory drugs (NSAIDs) in surface waters and tap water. Pristine and functionalised CNTs were evaluated as sorbent materials. Batch experiments were used to optimise sorption and desorption conditions (sorbent type and amount, adsorption time, pH). The adsorption equilibrium was reached after 8 to 48 h duration, which increased with the pH of solution. Non-agglomerated pristine CNTs (20 mg) showed the most optimal adsorption (94 to 100%) for all of the analytes after a 30-min contact period in acidified water solutions (100 mL). The compounds retained at those conditions were recovered by 40 to 95% by using 5% ammonium hydroxide in methanol as the desorbing solution at ambient conditions. A comprehensive liquid chromatography coupled to triple quadrupole mass spectrometry (LC-QqQ-MS/MS) was used for the analysis of real water samples. The method showed sufficient recovery (65-125%) and good precision (2-14% relative standard deviation (RSD)). The limits of detection and quantification ranged between 0.01 and 1.3 ng L-1 and 0.04 and 3.9 ng L-1. Only diclofenac and ibuprofen were found in the analysed surface water samples from Latvia (n = 10) and Norway (n = 14). Diclofenac was found at 1.7-8.4 ng L-1 concentration in two samples of surface waters, whereas the concentrations of ibuprofen ranged between 1.0 and 9.2 ng L-1 in seven samples collected in Norway and 3.9-17 ng L-1 in three samples from Latvia.

Determination of pharmaceutical residues and assessment of their removal efficiency at the Daugavgriva municipal wastewater treatment plant in Riga, Latvia.

Pharmaceutical products (PPs) belong to emerging contaminants that may accumulate along with other chemical pollutants in wastewaters (WWs) entering industrial and/or urban wastewater treatment plants (WWTPs). In the present study, the technique of ultra-high-performance liquid chromatography coupled to Orbitrap high-resolution mass spectrometry (Orbitrap-HRMS) was applied for the analysis of 24 multi-class PPs in WW samples collected at different technological stages of Daugavgriva WWTP located in Riga, Latvia. Caffeine and acetaminophen levels in the range of 7,570-11,403 ng/L and 810-1,883 ng/L, respectively, were the predominant compounds among 19 PPs determined in the WW. The results indicate that aerobic digestion in biological ponds was insufficiently effective to degrade most of the PPs (reduction efficiency <0-50.0%) with the exception of four PPs that showed degradation efficiency varying from 55.0 to 99.9%. Tests of short-term chemical and enzymatic hydrolysis for PP degradation in WW samples were performed, and the results reflected the complexity of different degradation mechanisms and physicochemical transformations of PPs. The toxicological studies of WW impact on Daphnia magna indicated gradual reduction of the total toxicity through the treatment stages at the WWTP.

17-beta-Estradiol stimulation of endothelial K+ channels.

Whole-cell membrane current recordings under voltage clamp were made from freshly isolated rabbit aortic endothelial cells. Intracellular Ca2+ signals of fura-2 loaded both native single aortic endothelial cells and intact endothelium from rabbit cardiac valves were measured using imaging fluorescence microscopy. Application of 17-beta-estradiol stimulated rapid discharge of transient outward currents and induced an increase in the intracellular free Ca2+ concentration ([Ca2+]i. The stimulated discharge of outward currents and [Ca2+]i were reduced by extracellulary applied tetraethylammonium (TEA).

Regenerative caffeine-induced responses in native rabbit aortic endothelial cells.

1. Single native aortic endothelial cells obtained by enzymatic dispersion of the rabbit aortic endothelium were held under voltage clamp using patch pipette and whole-cell membrane currents were measured. In parallel experiments performed on cells from the same batches, the free internal calcium concentration, [Ca2+]i, in the cell was estimated by use of the Ca(2+)-sensitive fluorescent dye, fura-2. 2. Caffeine (20 mM) applied to the cell evoked an outward current and an initial peak in [Ca2+]i followed by a lower sustained rise (plateau). Ca(2+)-free, EGTA-containing solution applied outside the cells did not reduce these responses. 3. Following caffeine stimulation there was a biphasic rising phase of outward current both in the presence and absence of extracellular Ca2+. 4. Application of graded doses of caffeine revealed all-or-none type responses of both the outward current and the rise in [Ca2+]i. 5. Preincubation with lower doses of caffeine reduced the magnitude of both the outward current and the [Ca2+]i transient evoked by 20 mM caffeine. 6. Tetraethylammonium (3 mM) applied to the bathing solution blocked unitary and spontaneous transient outward currents (STOCs) stimulated by Ca(2+)-free solution, but only reduced the outward current evoked by caffeine (20 mM). 7. In conclusion, our results reveal the all-or-none nature of Ca2+ release from the endoplasmic reticulum (ER) in native aortic endothelial cells. Lower concentrations of caffeine (0.4-0.5 mM) may deplete intracellular Ca2+ stores. Extracellular Ca2+ is not necessary for maintaining the activity of spontaneous and caffeine-induced outward currents in native aortic endothelial cells. Spontaneous outward currents are believed to represent the sporadic release of calcium from store sites independent of both extracellular Ca2+ and the caffeine-sensitive Ca2+ stores which stimulate the outward current.

Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.

1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current. 6. Externally applied tetraethylammonium ions (TEA, 3-10mM) reversibly inhibited unitary Ca2+-activated K+ currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine.7. Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine-evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine-induced outward current. Bath application of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evoked outward current in endothelial cells.8. Caffeine-induced Ca2+ release from intracellular stores evokes a transient rise in [Ca2+1, which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine sensitive current by TEA and Zn2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.

TEA inhibits ACh-induced EDRF release: endothelial Ca(2+)-dependent K+ channels contribute to vascular tone.

The effects of K(+)-channel blockers on the acetylcholine (ACh)-induced relaxation of vascular smooth muscle, intracellular free Ca2+ concentration ([Ca2+]i) elevation, and ACh-evoked outward K+ current of endothelial cells of rabbit aorta were studied using bioassay, spectrofluorimetry, and patch-clamp techniques, respectively. In bioassay experiments, ACh caused relaxation of endothelium-denuded aortic rings in a concentration-dependent manner when perfused through an endothelium-intact donor segment of aorta but not when perfused directly onto the recipient aortic ring. ACh-induced relaxation was inhibited by perfusion of tetraethylammonium ions (TEA; 5 mM) through the donor but not by perfusion directly onto the recipient segment. Glibenclamide had no effect on ACh-induced relaxation of the bioassay ring in either situation. ACh increased [Ca2+]i at the endothelial surface of aortic strips but not at the adventitial surface. TEA inhibited ACh-induced [Ca2+]i elevation, whereas glibenclamide had no effect. In patch-clamp experiments with freshly isolated endothelial cells, ACh evoked a biphasic outward current which was completely abolished by TEA (3 mM). It is concluded that Ca(2+)-dependent K+ channels are important for increasing [Ca2+]i during agonist stimulation and consequently for the synthesis/release of endothelium-derived relaxing factors (EDRFs). Furthermore, endothelial ATP-sensitive K+ channels do not contribute to ACh-induced relaxation or evoke an increase in endothelial [Ca2+]i of rabbit thoracic aorta.

Calcium-activated potassium channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block.

1. Isolated native endothelial cells, obtained by treatment of rabbit aortic endothelium with papain and dithiothreitol, were voltage clamped, and single channel (unitary) and spontaneous transient outward currents (STOCs) were recorded from both whole cells and excised membrane patches. 2. In inside-out patches, the reversal potential of unitary currents was dependent on the extracellular K+ concentration and had a single-channel slope conductance of 220 pS in symmetrical 140 mM-K+ solutions. The open-state probability (Po) of the unitary K+ currents was sensitive to the intracellular Ca2+ concentration with half-maximal activation at approximately 1 microM at +20 mV. The ionic selectivity and Ca2+ sensitivity indicate that a large conductance, Ca(2+)-activated K+ channel is present in freshly dissociated rabbit aortic endothelial cells. 3. The frequency and amplitude of whole-cell unitary currents and amplitude of spontaneous transient outward currents were voltage-dependent. Whole-cell outward K+ currents evoked by depolarizing voltage ramps had amplitudes often corresponding to the simultaneous opening of more than five single Ca(2+)-activated K+ channels. Lowering the intracellular EGTA concentration tenfold, and hence the Ca2+ buffering capacity of the cell, increased unitary K+ current activity and shifted the relationship between Po and membrane potential by approximately -20 mV. 4. Bradykinin (1 microM), adenosine 5'-triphosphate (3 microM) and acetylcholine (3 microM) applied extracellularly evoked a biphasic increase in N Po (where N is number of channels activated) of the Ca(2+)-activated K+ channel studied in the whole-cell recording configuration. The development of a biphasic response to agonist stimulation requires a source of extracellular Ca2+. The sustained increase in N Po of the Ca(2+)-activated K+ channel was attenuated upon the removal of external Ca2+ (Mg2+ replacement) or in the presence of the Ca2+ entry blocker, Ni2+, and the potassium channel blockers tetrabutylammonium (TBA) or tetraethylammonium (TEA). 5. Unitary and spontaneous transient outward currents were inhibited by extracellularly applied TEA (0.5 mM), TBA (0.5-5 mM) and charybdotoxin (100 nM). Ca(2+)-activated K+ currents were blocked completely by 5 mM-TEA, whereas 3,4-diaminopyridine (1 mM), Ba2+ (10 mM) and apamin (0.1-1 microM) did not abolish these K+ currents. 6. The K+ channel opener cromakalim (10 microM) evoked a sustained increase in N Po of the Ca(2+)-activated K+ channels which was not potentiated by the addition of bradykinin. Glibenclamide (10 microM) alone increased N Po and partially inhibited the cromakalim-induced increase in N Po with respect to control.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of phenylephrine in single isolated smooth muscle cells of rabbit and guinea pig taenia caeci.

Spontaneous transient and evoked outward currents were studied using the whole cell patch-clamp technique with freshly dispersed smooth muscle cells isolated from rabbit and guinea pig taenia caeci. Phenylephrine induced a low amplitude sustained outward current in both tissues. Simultaneously, the frequency and amplitude of spontaneous transient outward currents were increased. However, there were differences between the effects of phenylephrine on the evoked outward currents recorded from smooth muscle cells of rabbit (reduction) and guinea pig (enhancement) taenia caeci.

Calcium and the activation of the alpha 1-adrenoceptors in the guinea-pig taenia caeci.

1. The actions of phenylephrine (0.1-100 mumol l-1) and methoxamine (0.1-100 mumol l-1) were compared with that of adrenaline (0.01-10 mumol l-1) using the single sucrose gap method and mechanical recording in the guinea-pig taenia caeci. Drugs were applied for variable periods of time. 2. The characteristics of the inhibitory effects of alpha-adrenoceptor agonists were the same when exposure time did not exceed 5 min. When the exposure was prolonged, in contrast to the sustained effects of adrenaline (0.1-3 mumol l-1), phenylephrine and methoxamine (1-10 mumol l-1) produced a transient inhibitory action. 3. During the delayed recovery phase of phenylephrine, adrenaline preserved its ability to suppress the spontaneous electrical and mechanical activities of the taenia both when phenylephrine was replaced by adrenaline or when adrenaline was applied in addition to phenylephrine. All the above effects were found in untreated preparations, as well as during blockade of muscarinic cholinoceptors by atropine (1.4 mumol l-1), beta-adrenoceptors by propranolol (3 mumol l-1) and release of endogenous catecholamines by guanethidine (2.5 mumol l-1). 4. In the presence of phorbol 12,13-dibutyrate adrenaline ceased to be effective, while the inhibitory action of phenylephrine was converted to a contraction. 5. In calcium-free conditions in the presence of EGTA (0.4 mmol l-1) the initial hyperpolarization induced by adrenaline and phenylephrine was significantly reduced and with repeated applications of the agonists the inhibitory response disappeared. Similar results were obtained using tissues treated with nifedipine (1 and 10 mumol l-1). When caffeine (30mmolI 1) was present in the calciumfree solution the alpha-agonists studied were unable to produce any membrane potential changes. 6. The present results imply that the inhibitory effect of alpha-adrenoceptor agonists is mediated by the opening of potassium channels, which are activated by calcium derived from an intercellular source supplied from the extracellular space via a nifedipine-sensitive mechanism.

Effect of calcium entry blockade on the actions of phenylephrine on the taenia of the guinea pig caecum.

The interaction between phenylephrine and calcium entry blockers was studied on the taenia of the guinea-pig caecum using the double sucrose gap method. Sustained hyperpolarization, relaxation and attenuation of evoked electrical and mechanical activity were induced by non-cumulative addition of phenylephrine (0.1 to 250 mumol.1-1) for 2 to 4 min. When the alpha 1-adrenoceptor agonist was applied for a prolonged period (20 to 60 min) the initial inhibitory response gradually disappeared both at room temperature and at 32 degrees C. The renewed action potentials were accompanied by a positive afterpotential. The initial hyperpolarization and its delayed recovery in course of the phenylephrine effect were significantly reduced in calcium-free medium containing EDTA (2 mmol.1-1), after pretreatment with nifedipine (0.1 to 1 mumol.1-1), verapamil (10 to 100 mumol.1-1) or procaine (0.5 to 2 mmol.1-1). In contrast sodium nitroprusside (10 to 100 mumol.1-1) which produced biphasic changes similar to those of phenylephrine, did not affect the initial and delayed phase of phenylephrine action. Ba2+(5 mmol.1-1) could substitute for Ca2+ in the generation of action potentials but could not substitute for Ca2+ in the mechanisms responsible for the initial and delayed recovery phase of phenylephrine effects. In the presence of La3+ and Mn2+ (0.5 to 3 mmol.1-1) the phenylephrine effects were reduced. In contrast, in the presence of extracellular Ca2+, pretreatment with Mg2+ (12 mmol.1-1) or Ba2+ (5 mmol.1-1) did not affect the action of phenylephrine. It is concluded that activation of alpha 1-adrenoceptors results in the release of Ca2+ from an intracellular store, which leads to the opening od TEA-sensitive potassium channels, causing the initial phase of alpha 1-adrenoceptor action. Ca2+ is loaded into this intracellular store by entering the cell through the potential sensitive calcium channels. Although the mechanisms responsible for the delayed phase could not be clarified, its dependence on the presence of the initial phase is apparent.