Simultaneous measurement of intracellular Ca(2+) and nitric oxide: a new method

Different methods to measure the unstable radical nitric oxide (NO) have been established. We are going to present a new method to measure intracellular calcium and NO simultaneously in endothelial cells. A new fluorescent dye (DAF-2) has been developed recently which binds NO resulting in an enhanced fluorescence. We loaded porcine aortic endothelial cells with Fura-2, a fluorescent dye commonly used to measure intracellular calcium, and DAF-2 simultaneously (cell permeable dyes). Using excitation wavelengths of lambda 340 nm (Fura-2) and lambda 485 nm (DAF-2) we could show that thrombin induces an intracellular calcium increase and simultaneously a NO formation in endothelial cells which could be blocked by a NO synthase inhibitor. This new method of a simultaneous measurement of intracellular calcium and NO provides the possibility to follow intracellular calcium and NO distributions online, and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NO-synthase.     

Switched single-electrode voltage-clamp amplifiers allow precise measurement of gap junction conductance

Measurement of gapjunction conductance(g_j) with patch-clampamplifiers can, due to series resistance problems, be subject toconsiderable errors when large currents are measured. Formulas developed to correct for these errors unfortunately depend on exactestimates of series resistance, which are not always easy to obtain.Discontinuous single-electrode voltage-clamp amplifiers (DSEVCs) wereshown to overcome series resistance problems in single whole cellrecording. With the use of two synchronized DSEVCs, the simulatedg_j in a modelcircuit can be measured with a maximum error of <5% in all recordingsituations investigated (series resistance, 5-47 M; membraneresistance, 20-1,000 M; g_j, 1-100nS). At a very lowg_j of 100 pS, theerror sometimes exceeded 5% (maximum of 15%), but the error wasalways <5% when membrane resistance was >100 M. The precisionof the measurements is independent of series resistance, membraneresistance, and g_j. Consequently,it is possible to calculateg_j directly from Ohm's law, i.e., without using correction formulas. Our results suggest that DSEVCs should be used to measureg_j if largecurrents must be recorded, i.e., if cells are well coupled or ifmembrane resistance is low.     

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase-B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.     

Reaction rate constants of superoxide scavenging by plant antioxidants

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce. We describe an improved lucigenin-based chemiluminescence assay for kinetic analysis. The use of potassium superoxide (KO₂) as a nonenzymatic superoxide source allowed simple and reliable determination of the second-order reaction rate constants between superoxide and plant antioxidants at physiologically relevant conditions, avoiding unspecific effects of other reactive oxygen species or superoxide-generating enzymes. We calculated the rate constants for phenols of different structures, ranging from 2.9 × 10³ mol⁻¹ l s⁻¹ for morin to 2.9 × 10⁷ mol⁻¹ l s⁻¹ for proanthocyanidins. Compounds with pyrogallol or catechol moieties were revealed as the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure for maximal reaction rate constants with superoxide.     

A new method to measure nitrate/nitrite with a NO-sensitive electrode

There are different methods to measure the unstable moleculenitric oxide (NO). We will describe a new sensitive method to measure NO by reconversion of nitrate/nitrite to NO, which will bedetermined with an amperometric Clark-type electrode. Nitrate andnitrite are the degradation products of NO. First, nitrate isenzymatically converted to nitrite with the use of the nitrate reductase. Second, nitrite is reduced to equimolar NO concentrations byan acidic iodide solution. The detection limit of the electrode in anaqueous solution was 2 nmol/l NO (meaning the threshold was dependingon the volume added: 500 µl of a 0.2 µmol/l nitrite solution addedto a 10-ml bath). This method provides the ability to assess basal andagonist-stimulated NO releases of different biological models. Wemeasured basal and carbachol-stimulated NO release of nativeendothelial cells from porcine coronary arteries and porcine aorticendothelial cell cultures. Moreover, it was possible to measure thenitrate/nitrite concentration in the coronary effluent of a guinea pigheart. In conclusion, we present a valid, highly sensitive new methodof measuring nitrite/NO in biological systems with a commerciallyavailable electrode.